Petra Popovics - Profile on Academia.edu (original) (raw)

Papers by Petra Popovics

Proceedings of the National Academy of Sciences, 2013

Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to... more Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to the course of systemic inflammatory response syndrome or sepsis. The underlying mechanisms, however, are not well understood. Initial activation of adrenocortical hormone production during early sepsis depends on the stimulation of hypothalamus and pituitary mediated by cytokines; in late sepsis, there is a shift from neuroendocrine to local immuneadrenal regulation of glucocorticoid production. Therefore, the modulation of the local immune-adrenal cross talk, and not of the neuroendocrine circuits involved in adrenocorticotropic hormone production, may be more promising in the prevention of the adrenal insufficiency associated with prolonged sepsis. In the present work, we investigated the function of the crucial Toll-like receptor (TLR) adaptor protein myeloid differentiation factor 88 (MyD88) in systemic and local activation of adrenal gland inflammation and glucocorticoid production mediated by lipopolysachharides (LPSs). To this end, we used mice with a conditional MyD88 allele. These mice either were interbred with Mx1 Cre mice, resulting in systemic MyD88 deletion, predominantly in the liver and hematopoietic system, or were crossed with Akr1b7 Cre transgenic mice, resulting thereby in deletion of MyD88, which was adrenocortical-specific. Although reduced adrenal inflammation and HPAaxis activation mediated by LPS were found in Mx1 Cre+ -MyD88 fl/fl mice, adrenocortical-specific MyD88 deletion did not alter the adrenal inflammation or HPA-axis activity under systemic inflammatory response syndrome conditions. Thus, our data suggest an important role of immune cell rather than adrenocortical MyD88 for adrenal inflammation and HPA-axis activation mediated by LPS.

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth

Histochemistry and cell biology, Jan 15, 2015

Neurite growth is central to the formation and differentiation of functional neurons, and recentl... more Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compa...

Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocin-induced NOD/SCID mice

Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2015

Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote grow... more Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote growth, function, and engraftment of islet cells following transplantation. Here we evaluated recently synthesized GHRH agonists on the proliferation and biological functions of rat pancreatic β-cell line (INS-1) and islets. In vitro treatment of INS-1 cells with GHRH agonists increased cell proliferation, the expression of cellular insulin, insulin-like growth factor-1 (IGF1), and GHRH receptor, and also stimulated insulin secretion in response to glucose challenge. Exposure of INS-1 cells to GHRH agonists, MR-356 and MR-409, induced activation of ERK and AKT pathways. Agonist MR-409 also significantly increased the levels of cellular cAMP and the phosphorylation of cAMP response element binding protein (CREB) in INS-1 cells. Treatment of rat islets with agonist, MR-409 significantly increased cell proliferation, islet size, and the expression of insulin. In vivo daily s.c. administration o...

Oncotarget, Jan 14, 2015

We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective f... more We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair. H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF-α compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and...

Targeting the 5'-AMP-activated protein kinase and related metabolic pathways for the treatment of prostate cancer

INTRODUCTION: Increasing evidence suggests that prostate cancer cells undergo unique metabolic r... more INTRODUCTION:

Increasing evidence suggests that prostate cancer cells undergo unique metabolic reprogramming during transformation. A master regulator of cellular homeostasis, 5'-AMP-activated protein kinase (AMPK), directs metabolic adaptation that supports the growth demands of rapidly dividing cancer cells. The utilization of AMPK as a therapeutic target may therefore provide an effective strategy in the treatment of prostate cancer.
AREAS COVERED:

Our review describes the regulation of AMPK by androgens and upstream kinases including the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in prostate cancer. Oncogenic, AMPK-regulated pathways that direct various metabolic processes are also addressed. Furthermore, we discuss the role of AMPK in growth arrest and autophagy as a potential survival pathway for cancer cells. In addition, by regulating non-metabolic pathways, AMPK may stimulate migration and mitosis. Finally, this review summarizes efforts to treat prostate cancer with pharmacological agents capable of modulating AMPK signaling.
EXPERT OPINION:

Current research is primarily focused on developing drugs that activate AMPK as a treatment for prostate cancer. However, oncogenic aspects of AMPK signaling calls for caution about employing such therapies. We think that inhibitors of CaMKK2 or AMPK, or perhaps the modulation of downstream targets of AMPK, will gain importance in the clinical management of prostate cancer.

http://www.tandfonline.com/doi/full/10.4161/15384101.2015.945879

Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent ... more Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitro using the MTS assay. The effect of MIA-690 (5 μg/day 28 d) was further evaluated in vivo in nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 μM, and 19.2% at 5 μM, P < 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P < 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P < 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitro and in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.

Synthesis of new potent agonistic analogs of growth hormone-releasing hormone (GHRH) and evaluation of their endocrine and cardiac activities

Peptides, Feb 2014

In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on... more In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr(1)-JI-38), MR-361(N-Me-Tyr(1), D-Ala(2)-JI-38) and MR-367(N-Me-Tyr(1), D-Ala(2), Asn(8)-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr(1), showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr(1) and Arg(29)-NHCH3 as in MR-403 (N-Me-Tyr(1), D-Ala(2), Arg(29)-NHCH3-JI-38), MR-406 (N-Me-Tyr(1), Arg(29)-NHCH3-JI-38) and MR-409 (N-Me-Tyr(1), D-Ala(2), Asn(8), Arg(29)-NHCH3-JI-38), and MR-410 (N-Me-Tyr(1), D-Ala(2), Thr(8), Arg(29)-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa(30)-NH2 such as MR-326 (N-Me-Tyr(1), D-Ala(2), Arg(29), Apa(30)-NH2-JI-38), and with Gab(30)-NH2, as MR-502 (D-Ala(2), 5F-Phe(6), Ser(28), Arg(29),Gab(30)-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.

Oncoscience. 2014 October 30; 1(10):665 -673.

Introduction: This study evaluated the effects of an ntagonistic analog of growth hormone-releasi... more Introduction: This study evaluated the effects of an ntagonistic analog of growth hormone-releasing hormone, MIA-602, on tumor growth, response to doxorubicin, expression of drug resistance genes, and efflux pump function in human triple negative breast cancers.
Methods: HCC1806 (doxorubicin-sensitive) and MX-1 doxorubicin-resistant), cell lines were xenografted into nude mice and treated with MIA-602, doxorubicin, or their combination. Tumors were evaluated for changes in volume and the expression of the drug resistance genes MDR1 and NANOG. In-vitro cell culture assays were used to analyze the effect of MIA-602 on efflux pump function.
Results: Therapy with MIA-602 significantly reduced tumor growth and enhanced the efficacy of doxorubicin in both cell lines. Control HCC1806 tumors grew by 435%, while the volume of tumors treated with MIA-602 enlarged by 172.2% and with doxorubicin by 201.6%. Treatment with the combination of MIA-602 and doxorubicin resulted in an increase in volume of only 76.2%. Control MX-1 tumors grew by 907%, while tumors treated with MIA-602 enlarged by 434.8% and with doxorubicin by 815%. The combination of MIA-602 and doxorubicin reduced the increase in tumor volume to 256%. Treatment with MIA-602 lowered the level of growth hormonereleasing hormone and growth hormone-releasing hormone receptors and significantly reduced the expression of multidrug resistance (MDR1) gene and the drug resistance
regulator NANOG. MIA-602 also suppressed efflux pump function in both cell lines.
Conclusions: We conclude that treatment of triple negative breast cancers with growth hormone-releasing hormone antagonists reduces tumor growth and potentiates the effects of cytotoxic therapy by nullifying drug resistance.

World J Clin Urol. 2014 November 24; 3(3): 184-194. , Nov 24, 2014

Benign prostatic hyperplasia (BPH) is a pathologic condition of the prostate described as a subst... more Benign prostatic hyperplasia (BPH) is a pathologic condition of the prostate described as a substantial increase in its number of epithelial and stromal cells. BPH may significantly reduce the quality of life due to the initiation of bladder outlet obstruction and lower urinary tract syndromes. Current medical therapies mostly consist of inhibitors of 5α-reductase or α1-adrenergic blockers; their efficacy is often insufficient. Antagonistic analogs of neuropeptide hormones are novel candidates for the management of BPH. At first, antagonists of luteinizing hormone-releasing hormone (LHRH) have been introduced to the therapy aimed to reduce serum testosterone levels. However, they have also been found to produce an inhibitory activity on local LHRH receptors in the prostate as well as impotence and other related side effects. Since then, several preclinical and clinical studies reported the favorable effects
of LHRH antagonists in BPH. In contrast, antagonists of growth hormone-releasing hormone (GHRH) and gastrin-releasing peptide (GRP) have been tested only in preclinical settings and produce significant reduction in prostate size in experimental models of BPH. They act at least in part, by blocking the action of respective ligands produced locally on prostates through their respective receptors in the prostate, and by inhibition
of autocrine insulin-like growth factors-Ⅰ/Ⅱ and epidermal growth factor production. GHRH and LHRH antagonists were also tested in combination resulting in a cumulative effect that was greater than that of each alone. This article will review the numerous studies that demonstrate the beneficial effects of antagonistic analogs of LHRH, GHRH and GRP in BPH, as well as
suggesting a potential role for somatostatin analogs in experimental therapies.

Journal of cellular biochemistry, 2013

Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing... more Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ∼1 µM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca2+. The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ∼1 µM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca2+. The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Proceedings of the National Academy of Sciences of the United States of America, 2014

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities... more The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and geneexpression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and antiinvasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.

Proceedings of the National Academy of Sciences of the United States of America, 2013

Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to... more Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to the course of systemic inflammatory response syndrome or sepsis. The underlying mechanisms, however, are not well understood. Initial activation of adrenocortical hormone production during early sepsis depends on the stimulation of hypothalamus and pituitary mediated by cytokines; in late sepsis, there is a shift from neuroendocrine to local immuneadrenal regulation of glucocorticoid production. Therefore, the modulation of the local immune-adrenal cross talk, and not of the neuroendocrine circuits involved in adrenocorticotropic hormone production, may be more promising in the prevention of the adrenal insufficiency associated with prolonged sepsis. In the present work, we investigated the function of the crucial Toll-like receptor (TLR) adaptor protein myeloid differentiation factor 88 (MyD88) in systemic and local activation of adrenal gland inflammation and glucocorticoid production mediated by lipopolysachharides (LPSs). To this end, we used mice with a conditional MyD88 allele. These mice either were interbred with Mx1 Cre mice, resulting in systemic MyD88 deletion, predominantly in the liver and hematopoietic system, or were crossed with Akr1b7 Cre transgenic mice, resulting thereby in deletion of MyD88, which was adrenocortical-specific. Although reduced adrenal inflammation and HPAaxis activation mediated by LPS were found in Mx1 Cre+ -MyD88 fl/fl mice, adrenocortical-specific MyD88 deletion did not alter the adrenal inflammation or HPA-axis activity under systemic inflammatory response syndrome conditions. Thus, our data suggest an important role of immune cell rather than adrenocortical MyD88 for adrenal inflammation and HPA-axis activation mediated by LPS.

Peptides, 2013

In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on... more In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr 1 -JI-38), MR-361(N-Me-Tyr 1 , D-Ala 2 -JI-38) and MR-367(N-Me-Tyr 1 , D-Ala 2 , Asn 8 -JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr 1 , showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr 1 and Arg 29 -NHCH 3 as in MR-403 (N-Me-Tyr 1 , D-Ala 2 , Arg 29 -NHCH 3 -JI-38), MR-406 (N-Me-Tyr 1 , Arg 29 -NHCH 3 -JI-38) and MR-409 (N-Me-Tyr 1 , D-Ala 2 , Asn 8 , Arg 29 -NHCH 3 -JI-38), and MR-410 (N-Me-Tyr 1 , D-Ala 2 , Thr 8 , Arg 29 -NHCH 3 -JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa 30 -NH 2 such as MR-326 (N-Me-Tyr 1 , D-Ala 2 , Arg 29 , Apa 30 -NH 2 -JI-38), and with Gab 30 -NH 2 , as MR-502 (D-Ala 2 , 5F-Phe 6 , Ser 28 , Arg 29 ,Gab 30 -NH 2 -JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.

The Journal of endocrinology, 2011

Pituitary inhibin B, activin B, and follistatin are local regulators of FSH. Activin B is a homod... more Pituitary inhibin B, activin B, and follistatin are local regulators of FSH. Activin B is a homodimeric molecule (b B -b B ), while inhibin B contains an a and a b B subunit. The regulation of gene expression of a, b B , and follistatin by local and endocrine hormones was examined in pituitaries from female rats and in perifused pituitary cells by RT-PCR. Ovariectomy (OVX) induced an elevation in the mRNA level of a and b B subunits and follistatin. Short-term (4 h) treatment of pituitary cells with GnRH decreased both the inhibin a and the inhibin/activin b B subunit mRNA levels, while long-term treatment (20 h) with 100 nM GnRH stimulated the expression of both subunits. In contrast, the mRNA level of follistatin was elevated after the short-term GnRH treatment. Long-term exposure of pituitary cells to estradiol and inhibin B suppressed the mRNA expression of b B and had no effect on the expression of a subunit and follistatin. Our results demonstrate that the increased expressions of inhibin/activin subunits and follistatin in the post-OVX period can be induced by the lack of gonadal negative feedback, resulting in a high GnRH environment in the pituitary. This study reports for the first time that GnRH administered in high doses and for a long period stimulates the gene expression of inhibin/activin subunits and thereby may contribute to the stimulatory effect of OVX on the expression of these genes.

Phospholipase C-η2 is required for retinoic acid-stimulated neurite growth

Journal of neurochemistry, 2013

Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of... more Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca²⁺ signals by stimulating further Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLCη2 expression increased two-fold during a 4-day differentiation period. Stable expression of PLCη2-targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA-treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two-hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co-localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA-induced phosphorylation of LIMK1 and cAMP-responsive element-binding protein was reduced in PLCη2 knock-down cells. The phosphoinositide-binding properties of the PLCη2 PH domain, assessed using a FRET-based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P₃. Immunostaining of PLCη2 together with PtdIns(3,4,5)P₃ in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P₃ levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.

Phospholipase C-η activity may contribute to Alzheimer's disease-associated calciumopathy

Journal of Alzheimer's disease : JAD, 2012

Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) is assoc... more Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) is associated with altered neuronal Ca²⁺ homeostasis. Ca²⁺ is known to accumulate in AD-affected neurons leading to deficits in neurological activity that are characteristic of the disease. This has led to the coinage of the term &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;calciumopathy&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;. However, the mechanisms of how and why Ca²⁺ levels are increased in the AD-affected brain remain unknown. Identifying these mechanisms is crucial for our ability to treat and understand the disease processes that are occurring. Recent work has revealed the existence of a novel signaling pathway that may contribute toward this calciumopathy. Phospholipase C-η enzymes have recently been implicated in the modulation and amplification of Ca²⁺ signals and are known to be expressed in neuronal regions of the brain associated with cognition and memory. In this article their potential impact on neuronal Ca²⁺ signaling and AD pathogenesis is discussed.

Biochemical Society transactions, 2012

The most recently identified PLC (phospholipase C) enzymes belong to the PLCη family. Their uniqu... more The most recently identified PLC (phospholipase C) enzymes belong to the PLCη family. Their unique Ca 2 + -sensitivity and their specific appearance in neurons have attracted great attention since their discovery; however, their physiological role(s) in neurons are still yet to be established. PLCη enzymes are expressed in the neocortex, hippocampus and cerebellum. PLCη2 is also expressed at high levels in pituitary gland, pineal gland and in the retina. Driven by the specific localization of PLCη enzymes in different brain areas, in the present paper, we discuss the roles that they may play in neural processes, including differentiation, memory formation, circadian rhythm regulation, neurotransmitter/hormone release and the pathogenesis of neurodegenerative disorders associated with aberrant Ca 2 + signalling, such as Alzheimer's disease.

Cellular Signalling, 2011

Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely unch... more Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely uncharacterised. In this study the activity of PLCη2 was examined via [ 3 H]inositol phosphate release in COS7 cells expressing the enzyme. PLCη2 activity increased approximately 5-fold in response to monensin, a Na + /H + antiporter. This was significantly inhibited by CGP-37157 which implies that the effect of monensin was due, at least in part, to mitochondrial Na + /Ca 2+ -exchange. Direct activation of PLCη2 by b 1 μM Ca 2+ was confirmed in permeabilised transfected cells. The roles of the PH and C2 domains in controlling PLCη2 activity via membrane association were also investigated. A PH domain-lacking mutant exhibited no detectable activity in response to monensin or Ca 2+ due to an inability to associate with the cell membrane. Within the C2 domain, mutation of D920 to alanine at the predicted Ca 2+ -binding site dramatically reduced enzyme activity highlighting an important regulatory role for this domain. Mutation of D861 to asparagine also influenced activity, most likely due to altered lipid selectivity. Of the C2 mutations investigated, none altered sensitivity to Ca 2+ . This suggests that the C2 domain is not responsible for Ca 2+ activation. Collectively, this work highlights an important new component of the Ca 2+ signalling toolkit and given its sensitivity to Ca 2+ , this enzyme is likely to facilitate the amplification of intracellular Ca 2+ transients and/or crosstalk between Ca 2+ -storing compartments in vivo.

Experientia, 2011

GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subf... more GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn2+ has been shown to be a potent stimulator of GPR39 activity via the Gαq, Gα12/13 and Gαs pathways. The potency and specificity of Zn2+ in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn2+ signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.

Proceedings of the National Academy of Sciences, 2013

Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to... more Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to the course of systemic inflammatory response syndrome or sepsis. The underlying mechanisms, however, are not well understood. Initial activation of adrenocortical hormone production during early sepsis depends on the stimulation of hypothalamus and pituitary mediated by cytokines; in late sepsis, there is a shift from neuroendocrine to local immuneadrenal regulation of glucocorticoid production. Therefore, the modulation of the local immune-adrenal cross talk, and not of the neuroendocrine circuits involved in adrenocorticotropic hormone production, may be more promising in the prevention of the adrenal insufficiency associated with prolonged sepsis. In the present work, we investigated the function of the crucial Toll-like receptor (TLR) adaptor protein myeloid differentiation factor 88 (MyD88) in systemic and local activation of adrenal gland inflammation and glucocorticoid production mediated by lipopolysachharides (LPSs). To this end, we used mice with a conditional MyD88 allele. These mice either were interbred with Mx1 Cre mice, resulting in systemic MyD88 deletion, predominantly in the liver and hematopoietic system, or were crossed with Akr1b7 Cre transgenic mice, resulting thereby in deletion of MyD88, which was adrenocortical-specific. Although reduced adrenal inflammation and HPAaxis activation mediated by LPS were found in Mx1 Cre+ -MyD88 fl/fl mice, adrenocortical-specific MyD88 deletion did not alter the adrenal inflammation or HPA-axis activity under systemic inflammatory response syndrome conditions. Thus, our data suggest an important role of immune cell rather than adrenocortical MyD88 for adrenal inflammation and HPA-axis activation mediated by LPS.

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth

Histochemistry and cell biology, Jan 15, 2015

Neurite growth is central to the formation and differentiation of functional neurons, and recentl... more Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compa...

Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocin-induced NOD/SCID mice

Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2015

Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote grow... more Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote growth, function, and engraftment of islet cells following transplantation. Here we evaluated recently synthesized GHRH agonists on the proliferation and biological functions of rat pancreatic β-cell line (INS-1) and islets. In vitro treatment of INS-1 cells with GHRH agonists increased cell proliferation, the expression of cellular insulin, insulin-like growth factor-1 (IGF1), and GHRH receptor, and also stimulated insulin secretion in response to glucose challenge. Exposure of INS-1 cells to GHRH agonists, MR-356 and MR-409, induced activation of ERK and AKT pathways. Agonist MR-409 also significantly increased the levels of cellular cAMP and the phosphorylation of cAMP response element binding protein (CREB) in INS-1 cells. Treatment of rat islets with agonist, MR-409 significantly increased cell proliferation, islet size, and the expression of insulin. In vivo daily s.c. administration o...

Oncotarget, Jan 14, 2015

We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective f... more We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair. H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF-α compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and...

Targeting the 5'-AMP-activated protein kinase and related metabolic pathways for the treatment of prostate cancer

INTRODUCTION: Increasing evidence suggests that prostate cancer cells undergo unique metabolic r... more INTRODUCTION:

Increasing evidence suggests that prostate cancer cells undergo unique metabolic reprogramming during transformation. A master regulator of cellular homeostasis, 5'-AMP-activated protein kinase (AMPK), directs metabolic adaptation that supports the growth demands of rapidly dividing cancer cells. The utilization of AMPK as a therapeutic target may therefore provide an effective strategy in the treatment of prostate cancer.
AREAS COVERED:

Our review describes the regulation of AMPK by androgens and upstream kinases including the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in prostate cancer. Oncogenic, AMPK-regulated pathways that direct various metabolic processes are also addressed. Furthermore, we discuss the role of AMPK in growth arrest and autophagy as a potential survival pathway for cancer cells. In addition, by regulating non-metabolic pathways, AMPK may stimulate migration and mitosis. Finally, this review summarizes efforts to treat prostate cancer with pharmacological agents capable of modulating AMPK signaling.
EXPERT OPINION:

Current research is primarily focused on developing drugs that activate AMPK as a treatment for prostate cancer. However, oncogenic aspects of AMPK signaling calls for caution about employing such therapies. We think that inhibitors of CaMKK2 or AMPK, or perhaps the modulation of downstream targets of AMPK, will gain importance in the clinical management of prostate cancer.

http://www.tandfonline.com/doi/full/10.4161/15384101.2015.945879

Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent ... more Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitro using the MTS assay. The effect of MIA-690 (5 μg/day 28 d) was further evaluated in vivo in nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 μM, and 19.2% at 5 μM, P < 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P < 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P < 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitro and in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.

Synthesis of new potent agonistic analogs of growth hormone-releasing hormone (GHRH) and evaluation of their endocrine and cardiac activities

Peptides, Feb 2014

In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on... more In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr(1)-JI-38), MR-361(N-Me-Tyr(1), D-Ala(2)-JI-38) and MR-367(N-Me-Tyr(1), D-Ala(2), Asn(8)-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr(1), showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr(1) and Arg(29)-NHCH3 as in MR-403 (N-Me-Tyr(1), D-Ala(2), Arg(29)-NHCH3-JI-38), MR-406 (N-Me-Tyr(1), Arg(29)-NHCH3-JI-38) and MR-409 (N-Me-Tyr(1), D-Ala(2), Asn(8), Arg(29)-NHCH3-JI-38), and MR-410 (N-Me-Tyr(1), D-Ala(2), Thr(8), Arg(29)-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa(30)-NH2 such as MR-326 (N-Me-Tyr(1), D-Ala(2), Arg(29), Apa(30)-NH2-JI-38), and with Gab(30)-NH2, as MR-502 (D-Ala(2), 5F-Phe(6), Ser(28), Arg(29),Gab(30)-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.

Oncoscience. 2014 October 30; 1(10):665 -673.

Introduction: This study evaluated the effects of an ntagonistic analog of growth hormone-releasi... more Introduction: This study evaluated the effects of an ntagonistic analog of growth hormone-releasing hormone, MIA-602, on tumor growth, response to doxorubicin, expression of drug resistance genes, and efflux pump function in human triple negative breast cancers.
Methods: HCC1806 (doxorubicin-sensitive) and MX-1 doxorubicin-resistant), cell lines were xenografted into nude mice and treated with MIA-602, doxorubicin, or their combination. Tumors were evaluated for changes in volume and the expression of the drug resistance genes MDR1 and NANOG. In-vitro cell culture assays were used to analyze the effect of MIA-602 on efflux pump function.
Results: Therapy with MIA-602 significantly reduced tumor growth and enhanced the efficacy of doxorubicin in both cell lines. Control HCC1806 tumors grew by 435%, while the volume of tumors treated with MIA-602 enlarged by 172.2% and with doxorubicin by 201.6%. Treatment with the combination of MIA-602 and doxorubicin resulted in an increase in volume of only 76.2%. Control MX-1 tumors grew by 907%, while tumors treated with MIA-602 enlarged by 434.8% and with doxorubicin by 815%. The combination of MIA-602 and doxorubicin reduced the increase in tumor volume to 256%. Treatment with MIA-602 lowered the level of growth hormonereleasing hormone and growth hormone-releasing hormone receptors and significantly reduced the expression of multidrug resistance (MDR1) gene and the drug resistance
regulator NANOG. MIA-602 also suppressed efflux pump function in both cell lines.
Conclusions: We conclude that treatment of triple negative breast cancers with growth hormone-releasing hormone antagonists reduces tumor growth and potentiates the effects of cytotoxic therapy by nullifying drug resistance.

World J Clin Urol. 2014 November 24; 3(3): 184-194. , Nov 24, 2014

Benign prostatic hyperplasia (BPH) is a pathologic condition of the prostate described as a subst... more Benign prostatic hyperplasia (BPH) is a pathologic condition of the prostate described as a substantial increase in its number of epithelial and stromal cells. BPH may significantly reduce the quality of life due to the initiation of bladder outlet obstruction and lower urinary tract syndromes. Current medical therapies mostly consist of inhibitors of 5α-reductase or α1-adrenergic blockers; their efficacy is often insufficient. Antagonistic analogs of neuropeptide hormones are novel candidates for the management of BPH. At first, antagonists of luteinizing hormone-releasing hormone (LHRH) have been introduced to the therapy aimed to reduce serum testosterone levels. However, they have also been found to produce an inhibitory activity on local LHRH receptors in the prostate as well as impotence and other related side effects. Since then, several preclinical and clinical studies reported the favorable effects
of LHRH antagonists in BPH. In contrast, antagonists of growth hormone-releasing hormone (GHRH) and gastrin-releasing peptide (GRP) have been tested only in preclinical settings and produce significant reduction in prostate size in experimental models of BPH. They act at least in part, by blocking the action of respective ligands produced locally on prostates through their respective receptors in the prostate, and by inhibition
of autocrine insulin-like growth factors-Ⅰ/Ⅱ and epidermal growth factor production. GHRH and LHRH antagonists were also tested in combination resulting in a cumulative effect that was greater than that of each alone. This article will review the numerous studies that demonstrate the beneficial effects of antagonistic analogs of LHRH, GHRH and GRP in BPH, as well as
suggesting a potential role for somatostatin analogs in experimental therapies.

Journal of cellular biochemistry, 2013

Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing... more Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ∼1 µM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca2+. The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ∼1 µM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca2+. The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Proceedings of the National Academy of Sciences of the United States of America, 2014

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities... more The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and geneexpression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and antiinvasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.

Proceedings of the National Academy of Sciences of the United States of America, 2013

Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to... more Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to the course of systemic inflammatory response syndrome or sepsis. The underlying mechanisms, however, are not well understood. Initial activation of adrenocortical hormone production during early sepsis depends on the stimulation of hypothalamus and pituitary mediated by cytokines; in late sepsis, there is a shift from neuroendocrine to local immuneadrenal regulation of glucocorticoid production. Therefore, the modulation of the local immune-adrenal cross talk, and not of the neuroendocrine circuits involved in adrenocorticotropic hormone production, may be more promising in the prevention of the adrenal insufficiency associated with prolonged sepsis. In the present work, we investigated the function of the crucial Toll-like receptor (TLR) adaptor protein myeloid differentiation factor 88 (MyD88) in systemic and local activation of adrenal gland inflammation and glucocorticoid production mediated by lipopolysachharides (LPSs). To this end, we used mice with a conditional MyD88 allele. These mice either were interbred with Mx1 Cre mice, resulting in systemic MyD88 deletion, predominantly in the liver and hematopoietic system, or were crossed with Akr1b7 Cre transgenic mice, resulting thereby in deletion of MyD88, which was adrenocortical-specific. Although reduced adrenal inflammation and HPAaxis activation mediated by LPS were found in Mx1 Cre+ -MyD88 fl/fl mice, adrenocortical-specific MyD88 deletion did not alter the adrenal inflammation or HPA-axis activity under systemic inflammatory response syndrome conditions. Thus, our data suggest an important role of immune cell rather than adrenocortical MyD88 for adrenal inflammation and HPA-axis activation mediated by LPS.

Peptides, 2013

In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on... more In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr 1 -JI-38), MR-361(N-Me-Tyr 1 , D-Ala 2 -JI-38) and MR-367(N-Me-Tyr 1 , D-Ala 2 , Asn 8 -JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr 1 , showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr 1 and Arg 29 -NHCH 3 as in MR-403 (N-Me-Tyr 1 , D-Ala 2 , Arg 29 -NHCH 3 -JI-38), MR-406 (N-Me-Tyr 1 , Arg 29 -NHCH 3 -JI-38) and MR-409 (N-Me-Tyr 1 , D-Ala 2 , Asn 8 , Arg 29 -NHCH 3 -JI-38), and MR-410 (N-Me-Tyr 1 , D-Ala 2 , Thr 8 , Arg 29 -NHCH 3 -JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa 30 -NH 2 such as MR-326 (N-Me-Tyr 1 , D-Ala 2 , Arg 29 , Apa 30 -NH 2 -JI-38), and with Gab 30 -NH 2 , as MR-502 (D-Ala 2 , 5F-Phe 6 , Ser 28 , Arg 29 ,Gab 30 -NH 2 -JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.

The Journal of endocrinology, 2011

Pituitary inhibin B, activin B, and follistatin are local regulators of FSH. Activin B is a homod... more Pituitary inhibin B, activin B, and follistatin are local regulators of FSH. Activin B is a homodimeric molecule (b B -b B ), while inhibin B contains an a and a b B subunit. The regulation of gene expression of a, b B , and follistatin by local and endocrine hormones was examined in pituitaries from female rats and in perifused pituitary cells by RT-PCR. Ovariectomy (OVX) induced an elevation in the mRNA level of a and b B subunits and follistatin. Short-term (4 h) treatment of pituitary cells with GnRH decreased both the inhibin a and the inhibin/activin b B subunit mRNA levels, while long-term treatment (20 h) with 100 nM GnRH stimulated the expression of both subunits. In contrast, the mRNA level of follistatin was elevated after the short-term GnRH treatment. Long-term exposure of pituitary cells to estradiol and inhibin B suppressed the mRNA expression of b B and had no effect on the expression of a subunit and follistatin. Our results demonstrate that the increased expressions of inhibin/activin subunits and follistatin in the post-OVX period can be induced by the lack of gonadal negative feedback, resulting in a high GnRH environment in the pituitary. This study reports for the first time that GnRH administered in high doses and for a long period stimulates the gene expression of inhibin/activin subunits and thereby may contribute to the stimulatory effect of OVX on the expression of these genes.

Phospholipase C-η2 is required for retinoic acid-stimulated neurite growth

Journal of neurochemistry, 2013

Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of... more Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca²⁺ signals by stimulating further Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLCη2 expression increased two-fold during a 4-day differentiation period. Stable expression of PLCη2-targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA-treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two-hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co-localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA-induced phosphorylation of LIMK1 and cAMP-responsive element-binding protein was reduced in PLCη2 knock-down cells. The phosphoinositide-binding properties of the PLCη2 PH domain, assessed using a FRET-based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P₃. Immunostaining of PLCη2 together with PtdIns(3,4,5)P₃ in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P₃ levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.

Phospholipase C-η activity may contribute to Alzheimer's disease-associated calciumopathy

Journal of Alzheimer's disease : JAD, 2012

Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) is assoc... more Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (AD) is associated with altered neuronal Ca²⁺ homeostasis. Ca²⁺ is known to accumulate in AD-affected neurons leading to deficits in neurological activity that are characteristic of the disease. This has led to the coinage of the term &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;calciumopathy&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;. However, the mechanisms of how and why Ca²⁺ levels are increased in the AD-affected brain remain unknown. Identifying these mechanisms is crucial for our ability to treat and understand the disease processes that are occurring. Recent work has revealed the existence of a novel signaling pathway that may contribute toward this calciumopathy. Phospholipase C-η enzymes have recently been implicated in the modulation and amplification of Ca²⁺ signals and are known to be expressed in neuronal regions of the brain associated with cognition and memory. In this article their potential impact on neuronal Ca²⁺ signaling and AD pathogenesis is discussed.

Biochemical Society transactions, 2012

The most recently identified PLC (phospholipase C) enzymes belong to the PLCη family. Their uniqu... more The most recently identified PLC (phospholipase C) enzymes belong to the PLCη family. Their unique Ca 2 + -sensitivity and their specific appearance in neurons have attracted great attention since their discovery; however, their physiological role(s) in neurons are still yet to be established. PLCη enzymes are expressed in the neocortex, hippocampus and cerebellum. PLCη2 is also expressed at high levels in pituitary gland, pineal gland and in the retina. Driven by the specific localization of PLCη enzymes in different brain areas, in the present paper, we discuss the roles that they may play in neural processes, including differentiation, memory formation, circadian rhythm regulation, neurotransmitter/hormone release and the pathogenesis of neurodegenerative disorders associated with aberrant Ca 2 + signalling, such as Alzheimer's disease.

Cellular Signalling, 2011

Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely unch... more Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely uncharacterised. In this study the activity of PLCη2 was examined via [ 3 H]inositol phosphate release in COS7 cells expressing the enzyme. PLCη2 activity increased approximately 5-fold in response to monensin, a Na + /H + antiporter. This was significantly inhibited by CGP-37157 which implies that the effect of monensin was due, at least in part, to mitochondrial Na + /Ca 2+ -exchange. Direct activation of PLCη2 by b 1 μM Ca 2+ was confirmed in permeabilised transfected cells. The roles of the PH and C2 domains in controlling PLCη2 activity via membrane association were also investigated. A PH domain-lacking mutant exhibited no detectable activity in response to monensin or Ca 2+ due to an inability to associate with the cell membrane. Within the C2 domain, mutation of D920 to alanine at the predicted Ca 2+ -binding site dramatically reduced enzyme activity highlighting an important regulatory role for this domain. Mutation of D861 to asparagine also influenced activity, most likely due to altered lipid selectivity. Of the C2 mutations investigated, none altered sensitivity to Ca 2+ . This suggests that the C2 domain is not responsible for Ca 2+ activation. Collectively, this work highlights an important new component of the Ca 2+ signalling toolkit and given its sensitivity to Ca 2+ , this enzyme is likely to facilitate the amplification of intracellular Ca 2+ transients and/or crosstalk between Ca 2+ -storing compartments in vivo.

Experientia, 2011

GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subf... more GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn2+ has been shown to be a potent stimulator of GPR39 activity via the Gαq, Gα12/13 and Gαs pathways. The potency and specificity of Zn2+ in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn2+ signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.