Thomas Spelsberg - Academia.edu (original) (raw)

Papers by Thomas Spelsberg

Journal of Biological Chemistry, 2009

3 The abbreviations used are: T reg, CD4 ϩ CD25 ϩ regulatory T cells; TGF-␤1, transforming growth... more 3 The abbreviations used are: T reg, CD4 ϩ CD25 ϩ regulatory T cells; TGF-␤1, transforming growth factor-␤1; KLF10, Kruppel-like factor 10; EV, empty virus; ChIP, chromatin immunoprecipitation; WT, wild-type; SEAP, secreted form of human placental alkaline phosphatase; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; APC, antigen presenting cell; MIP, macrophage inflammatory protein; MCP, --; MMP, --; SDF, stromal cell derived factor; mAb, monoclonal antibody; IL, interleukin; IFN, interferon; RANTES, regulated on activation normal T cell expressed and secreted; TCR, T cell receptor; MCP, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase.

Journal of Biological Chemistry, 1997

The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previ... more The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-myc gene promoter. This element contains a 5-GC-rich domain and a 3-AT-rich domain, the latter of which has a homopurine/homopyrimidine structure. The gel shift assays required the generation of an RBF-maltose fusion protein (RBF-MBP), which specifically binds this element and is supershifted when the anti-RBF polyclonal antibody is added. Computer analysis of the fulllength amino acid sequence for RBF predicts a DNAbinding motif involving a ␤-sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-myc promoter, which flank an intervening domain containing the RBF element. The co-transfection of this MAR sequence, containing the RBF element and cloned into a luciferase reporter vector, together with an RBF expression vector construct, into steroid treated human MCF-7 cells, results in a decrease of the c-myc promoter activity relative to control transfections containing only the parent vector of the RBF expression construct. These data suggest that a unique chromatin/ nuclear matrix structure, composed of the RBF-DNA element complex which is flanked by nuclear matrix attachment sites, serves to bind the PR and repress the c-myc promoter.

Genomics, 1998

TGF␤-inducible early gene (TIEG) and early growth response ␣ (EGR␣) are putative transcription fa... more TGF␤-inducible early gene (TIEG) and early growth response ␣ (EGR␣) are putative transcription factors based on homology to known zinc finger proteins SP1, EGR1, BTEB, and Wilm tumor. Here we report that TIEG and EGR␣ are expressed from alternative promoters of the same gene. The TIEG/EGR␣ gene spans 8 kb and contains five exons. Use of alternative first exons results in TIEG having 12 unique amino acids on its N-terminus. Computer analysis of the 5 upstream regions of either TIEG (exon 1a) or EGR␣ (exon 1b) does not identify a TATA box or initiator sequence but shows consensus sequence similarities to binding sites for several transcription factors including SP1, JunB, and aromatic hydrocarbon/receptor-ligand complexes. Analysis of constructs containing 5-flanking regions show that both the TIEG and the EGR␣ promoters have significant activity in human fetal osteoblast cells. Northern analysis of mRNA from various human tissues and several cell lines reveals that TIEG is the predominant transcript produced and regulated by growth factors from the TIEG/EGR␣ gene.

Molecular and Cellular Biology, 2010

The circadian timing system coordinates many aspects of mammalian physiology and behavior in sync... more The circadian timing system coordinates many aspects of mammalian physiology and behavior in synchrony with the external light/dark cycle. These rhythms are driven by endogenous molecular clocks present in most body cells. Many clock outputs are transcriptional regulators, suggesting that clock genes primarily control physiology through indirect pathways. Here, we show that Krüppel-like factor 10 (KLF10) displays a robust circadian expression pattern in wild-type mouse liver but not in clock-deficient Bmal1 knockout mice. Consistently, the Klf10 promoter recruited the BMAL1 core clock protein and was transactivated by the CLOCK-BMAL1 heterodimer through a conserved E-box response element. Profiling the liver transcriptome from Klf10(-/-) mice identified 158 regulated genes with significant enrichment for transcripts involved in lipid and carbohydrate metabolism. Importantly, approximately 56% of these metabolic genes are clock controlled. Male Klf10(-/-) mice displayed postprandial and fasting hyperglycemia, a phenotype accompanied by a significant time-of-day-dependent upregulation of the gluconeogenic gene Pepck and increased hepatic glucose production. Consistently, functional data showed that the proximal Pepck promoter is repressed directly by KLF10. Klf10(-/-) females were normoglycemic but displayed higher plasma triglycerides. Correspondingly, rhythmic gene expression of components of the lipogenic pathway, including Srebp1c, Fas, and Elovl6, was altered in females. Collectively, these data establish KLF10 as a required circadian transcriptional regulator that links the molecular clock to energy metabolism in the liver.

Signal Transduction, 2004

Signaling through the Transforming Growth Factor-β (TGFβ)/Smad pathway plays an important role in... more Signaling through the Transforming Growth Factor-β (TGFβ)/Smad pathway plays an important role in a variety of cellular processes including the control of cellular proliferation and oncogenesis. The TGFβ Inducible Early Gene (TIEG) is a primary response gene for TGFβ which controls the activity of the Smad pathway. In addition, studies from our laboratory have demonstrated that TIEG expression is lost during the progression of breast cancer. In the present study we utilized a tetracycline inducible TIEG overexpressing breast cancer cell line and TIEG null mouse embryo fibroblasts (MEFs) to establish whether TIEG plays a central role in eliciting the anti-proliferative effects of TGFβ. We demonstrate that, similar to TGFβ treatment, TIEG overexpression significantly decreases cellular proliferation. Consistent with a role in regulating cellular proliferation, TIEG overexpression increases the expression of the cyclin dependent kinase inhibitor p21. Interestingly, while cellular proliferation of wild-type MEFs is inhibited by TGFβ, proliferation of TIEG null MEFs is stimulated by TGFβ. Furthermore, TIEG null MEF cells display a decrease in Smad dependent transcription with a concomitant prolonged increase in Smad7 expression compared to wild-type cells. These data strongly suggest that TIEG plays a central role in the anti-proliferative response to TGFβ and may explain how a loss of TIEG expression contributes to the development of cancer.

Proceedings of The National Academy of Sciences, 1999

For many years it has been recognized that sex steroids have profound effects on bone metabolism.... more For many years it has been recognized that sex steroids have profound effects on bone metabolism. The current perception is that estrogen decreases bone resorption and androgen increases bone deposition. To investigate the potential for androgens to directly modulate bone resorption, we have examined avian osteoclast and human and mouse osteoclast-like cells for androgen responsiveness. There was a dose-dependent decrease

Proceedings of The National Academy of Sciences, 1994

The decrease in estrogen levels that follows the onset of menopause results in rapid bone loss an... more The decrease in estrogen levels that follows the onset of menopause results in rapid bone loss and osteoporosis. The major effect of estrogen deficiency on bone metabolism is an increase in the rate of bone resorption, but the precise mechanism by which this occurs remains unresolved. A recently developed technique for the isolation of avian osteoclasts has been modified to

Estrogen is a major sex steroid that affects the growth, maintenance, and homeostasis of the skel... more Estrogen is a major sex steroid that affects the growth, maintenance, and homeostasis of the skeleton. Two iso- forms of the estrogen receptor (ER and ER) mediate the transcriptional effects of estrogen. Although both isoforms of ER are present and functional in some human osteoblast (OB) cell lines, there is minimal information on the differential regulation of transcription by ER

Molecular and cellular biology, 2005

Transforming growth factor beta-inducible early gene 1 (TIEG1) is a member of the Kruppel-like tr... more Transforming growth factor beta-inducible early gene 1 (TIEG1) is a member of the Kruppel-like transcription factor family. To understand the physiological role of TIEG1, we generated TIEG(-/-) (null) mice and found that the TIEG(-/-) mice had increased osteoblast numbers with no increased bone formation parameters. However, when calvarial osteoblasts (OBs) were isolated from neonatal TIEG(-/-) and TIEG(+/+) mice and cultured in vitro, the TIEG(-/-) cells displayed reduced expression of important OB differentiation markers. When the OBs were differentiated in vitro by treatment with bone morphogenic protein 2, the OBs from TIEG(+/+) calvaria displayed several mineralized nodules in culture, whereas those from TIEG(-/-) mice showed no nodules. To characterize the OBs' ability to support osteoclast differentiation, the OBs from TIEG(+/+) and TIEG(-/-) mice were cultured with marrow and spleen cells from TIEG(+/+) mice. Significantly fewer osteoclasts developed when TIEG(-/-) OBs w...

PloS one, 2014

Endoxifen has recently been identified as the predominant active metabolite of tamoxifen and is c... more Endoxifen has recently been identified as the predominant active metabolite of tamoxifen and is currently being developed as a novel hormonal therapy for the treatment of endocrine sensitive breast cancer. Based on past studies in breast cancer cells and model systems, endoxifen classically functions as an anti-estrogenic compound. Since estrogen and estrogen receptors play critical roles in mediating bone homeostasis, and endoxifen is currently being implemented as a novel breast cancer therapy, we sought to comprehensively characterize the in vivo effects of endoxifen on the mouse skeleton. Two month old ovariectomized C57BL/6 mice were treated with vehicle or 50 mg/kg/day endoxifen hydrochloride via oral gavage for 45 days. Animals were analyzed by dual-energy x-ray absorptiometry, peripheral quantitative computed tomography, micro-computed tomography and histomorphometry. Serum from control and endoxifen treated mice was evaluated for bone resorption and bone formation markers. ...

Bisphosphonates are widely used clinically to treat bone diseases in which bone resorption is in ... more Bisphosphonates are widely used clinically to treat bone diseases in which bone resorption is in excess. However, the mechanism of bisphos- phonate action on bone is not fully understood. Studies of direct action of bisphosphonates on bone have been limited mainly to their effects on bone-resorbing osteoclast cells, with implications that some activity may be mediated indirectly through paracrine factors

The Journal of Steroid Biochemistry and Molecular Biology, 2005

Certain plant-derived compounds show selective estrogen receptor modulator (SERM) activity and ma... more Certain plant-derived compounds show selective estrogen receptor modulator (SERM) activity and may therefore be an alternative to the conventional hormone replacement therapy, which prevents osteoporosis but is also associated with an increased risk of breast and endometrial cancers. In the current study, we tested the effects of the hop-derived compounds 8-prenylnaringenin, 6-prenylnaringenin, xanthohumol and isoxanthohumol (1) to modulate markers of differentiation and gene expression in osteoblasts and (2) to regulate proliferation in MCF-7 breast cancer cells. Additionally, we analyzed the ER-binding affinities of these hop compounds as well as the ER-mediation of their effects. Bone-forming activity and ER-subtype specificity were investigated by measuring alkaline phosphatase (AP) activity in hFOB/ERalpha cells and regulation of gene transcription for AP, interleukin-6, pS2 and von Willebrand factor (VWF) in U-2 OS/ERalpha and U-2 OS/ERbeta cells. Our results demonstrate that AP, pS2 and VWF mRNA levels are significantly increased by the compounds in an estrogen-like manner via both ERalpha and ERbeta, while IL-6 is down-regulated in U-2 OS/ERalpha cells. Consistently, AP enzymatic activity is up-regulated by all compounds in hFOB/ERalpha9 cells. Depending on their concentration, all compounds show proliferative effects in MCF-7 cells. Except for 8-PN the hop constituents display an ERbeta-preference. Reversal of estrogen-specific AP-induction in Ishikawa cells indicates an ER-regulated mechanism. Finally, the flavonoids display cytotoxic effects only at high concentrations (&amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; or =10(-4)M). In summary, we have demonstrated for the first time that specific phytoestrogen compounds found in hop extracts exert estrogen-like activities on bone metabolism. Regarding a potential for use in osteoporosis-prevention therapy, the dosage of a phytoestrogen, which is taken, will play an important role concerning a desired in vivo profile.

PLoS ONE, 2011

TGF-b Inducible Early Gene-1 (TIEG1) is a Krü ppel-like transcription factor (KLF10) that was ori... more TGF-b Inducible Early Gene-1 (TIEG1) is a Krü ppel-like transcription factor (KLF10) that was originally cloned from human osteoblasts as an early response gene to TGF-b treatment. As reported previously, TIEG1 2/2 mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1 2/2 osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1 2/2 precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1 2/2 osteoclasts. We observed increased AKT and MEK/ ERK signaling pathway activation in TIEG1 2/2 osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1) to TIEG1 2/2 cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1 2/2 precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling.

Oncogene, 2002

TGFbeta inducible early gene (TIEG) encodes a three zinc-finger Krüppel-like transcription factor... more TGFbeta inducible early gene (TIEG) encodes a three zinc-finger Krüppel-like transcription factor whose overexpression has been shown to mimic the effects of TGFbeta in human osteosarcoma and pancreatic carcinoma cells. In order to investigate a potential role of TIEG in the TGFbeta signal transduction pathway, we studied its impact on a Smad binding element (SBE) reporter which is known to be regulated by TGFbeta through the R-Smad proteins. We demonstrate that TIEG overexpression enhances TGFbeta induction of SBE reporter activity. TIEG overexpression also enhances induction of the endogenous TGFbeta regulated genes p21 and PAI-1. The ability of TIEG to enhance TGFbeta actions is Smad dependent since TIEG has no effect on SBE transcription in the absence of Smad4 expression or when an inhibitory Smad protein, Smad7, is overexpressed. Furthermore, TIEG overexpression enhances TGFbeta induced Smad2 phosphorylation. Lastly, TIEG appears to function by binding to and thereby repressing a specific element in the proximal promoter of the inhibitory Smad7 gene. In conclusion, these results describe a novel mechanism for the potentiation of TGFbeta/Smad signaling via repression of the inhibitory Smad7 gene by TIEG.

Nucleic Acids Research, 1995

Page 1. Nucleic Acids Research, 1995, Vol. 23, No. 23 4907-4912 Identification of a novel TGF-p-r... more Page 1. Nucleic Acids Research, 1995, Vol. 23, No. 23 4907-4912 Identification of a novel TGF-p-regulated gene encoding a putative zinc finger protein in human osteoblasts Malayannan Subramaniam*, Steven A. Harris, Merry ...

New England Journal of Medicine, 2003

a lower endocochlear potential than Cx30 +/¡ heterozygotes, our results indicate that not only Cx... more a lower endocochlear potential than Cx30 +/¡ heterozygotes, our results indicate that not only Cx30 (GJB6) 5 but also Cx26 (GJB2) is involved in the production of the endocochlear potential.

Molecular Endocrinology, 1999

... Accepted March 22, 1999. Previous Section. References. ↵ Riggs BL, Melton LJ 1995 Osteoporosi... more ... Accepted March 22, 1999. Previous Section. References. ↵ Riggs BL, Melton LJ 1995 Osteoporosis: Etiology, Diagnosis, and Management, ed 2. Lippincott-Raven Publishers, Philadelphia, PA. ↵ Bilezikian JP, Raisz LG, Rodan GA 1996 Principles of Bone Biology. ...

Molecular Endocrinology, 2008

The estrogen receptors (ER) ␣ and ␤ are important ligand-mediated transcription factors known to ... more The estrogen receptors (ER) ␣ and ␤ are important ligand-mediated transcription factors known to play significant biological roles in numerous tissues including bone. Despite the high homology shared by these receptors, recent studies have suggested that their function is largely unique. Although these receptors have been studied in detail for more than a decade, little data exist concerning the mechanisms by which these two proteins regulate distinct sets of genes. Using the TGF␤-inducible early gene-1 (TIEG) as a model, we demonstrate that TIEG is rapidly induced in response to estrogen in osteoblasts by ER␤, but not ER␣. We have identified the regulatory elements utilized by ER␤ and have demonstrated that ER␤ recruits steroid receptor coactivator (SRC)1 and SRC2 to this regulatory region. Additionally, deletion of the

Mayo Clinic Proceedings, 2002

Genomics has been defined as the comprehensive study of whole sets of genes, gene products, and t... more Genomics has been defined as the comprehensive study of whole sets of genes, gene products, and their interactions as opposed to the study of single genes or proteins. Microarray technology is one of many novel tools that are allowing global and high-throughput analysis of genes and gene products. In addition to an introduction on underlying principles, the current review focuses on the use of both complementary DNA and oligodeoxynucleotide microarrays in gene expression analysis. Genome-wide experiments generate a massive amount of data points that require systematic methods of analysis to extract biologically useful information. Accordingly, the current educational communication discusses different methods of data analysis, including supervised and unsupervised clustering algorithms. Illustrative clinical examples show clinical applications, including (1) identification of candidate genes or pathological pathways (ie, elucidation of pathogenesis); (2) identification of "new" molecular classes of diseases ALL = acute lymphocytic leukemia; AML = acute myeloid leukemia; bp = base pair; cDNA = complementary DNA; CLL = chronic lymphocytic leukemia; cRNA = complementary RNA; DLBCL = diffuse large B-cell lymphoma; EST = expressed sequence tag; FL = follicular lymphoma; mRNA = messenger RNA; PCR = polymerase chain reaction; SOM = self-organizing map that may be relevant in disease reclassification, prognostication, and treatment selection (ie, class discovery); and (3) use of expression profiles of known disease classes to predict diagnosis and classification of unknown samples (ie, class prediction). The current review should serve as an introduction to the subject for clinician investigators, physicians and medical scientists in training, practicing clinicians, and other students of medicine.