Mike Connor | York University (original) (raw)
Papers by Mike Connor
Nature Medicine, 2002
Cell-cycle deregulation is a hallmark of cancer. Loss of cytokinemediated G1 arrest may confer an... more Cell-cycle deregulation is a hallmark of cancer. Loss of cytokinemediated G1 arrest may confer an advantage during malignant progression. Resistance to the antiproliferative effects of transforming growth factor-β (TGF-β) often occurs despite intact TGFβ signaling and such cells may manifest resistance to multiple inhibitory cytokines, suggesting underlying alterations in cellcycle controls 1,2. Cyclin-dependent kinases (cdks) are regulated by cyclin binding, phosphorylation and by two families of cdk inhibitors 3. G1 progression is governed by D-type and E-type cyclin-cdk complexes. The inhibitors of cdk4 (INK4) family includes p15 INK4B and the kinase inhibitor protein (KIP) family comprises p21 Cip1 , p27 Kip1 and p57 Kip2 (ref. 4). In addition to inhibition of cyclin E-cdk2, p21 and p27 also facilitate assembly and activation of cyclin D-cdks in early G1 (refs. 5,6). p27 Kip1 was first identified as a mediator of TGF-β1-induced G1 arrest 7-9. TGF-β induces expression of the gene encoding p15 INK4B. p15 INK4B binds and inhibits cdk4 facilitating dissociation of p27 and cyclin D1 from cyclin D1-cdk4-p27 complexes and p27 binds and inhibits cyclin E-cdk2, leading to G1 arrest 10-12. p27 also mediates G1 arrest induced by IL-6 (ref. 13). p27 is a nuclear protein whose frequent deregulation in human cancers may confer resistance to antiproliferative signals. In cMyc or MAPK activated cancer-derived lines, cyclin D1-cdk4/6 complexes sequester p27, and cyclin E-cdk2-inhibition is impaired 14-16. cMyc inhibits p15 induction by TGF-β (ref. 17) and may also induce a factor that inactivates p27 18. In up to 50% of human cancers, reduced p27 protein is associated with a poor prognosis 19. In some tumors, p27 is mislocalized to the cytoplasm 19,20 , however; the mechanism and significance of this has not been elucidated. In human cancers, constitutive activation of phosphoinositol 3′ kinase (PI3K) and its effector PKB/Akt arise through oncogenic receptor tyrosine kinase activation, Ras activation, mutational loss of PTEN, or through activating mutation of the PI3K effector, protein kinase B (PKB)/Akt (hereafter termed Akt) itself 21,22. Akt can increase cyclin D1 levels 23 and downregulate p27 by increasing p27 proteolysis 24 or repressing p27 expression through Akt phosphorylation of a forkhead transcription factor 25. However, in most cancers, reduced p27 does not result from transcriptional silencing 19. Here we show that Akt causes resistance to cytokine-mediated G1 arrest. p27 phosphorylation by Akt impairs its nuclear import and leads to cytoplasmic p27 accumulation. In human breast cancers, cytoplasmic mislocalization of p27 is associated with Akt activation, loss of differentiation and poor patient outcome. Activation of Akt in lines resistant to G1 arrest by TGF-β Ras has been shown to confer TGF-β resistance. While investigating mechanisms of TGF-β resistance, we found that two TGF-β PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest
The Journal of Urology, 2012
American Journal of Physiology-Endocrinology and Metabolism, 1999
We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation ... more We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation and mRNA stability with unilateral chronic stimulation (10 Hz, 3 h/day) of the rat tibialis anterior (TA) muscle for 1, 2, 3, 4, 5, and 7 days ( n = 3–11/group). Transcriptional activation was assessed by direct plasmid DNA injection into the TA with a chloramphenicol acetyltransferase (CAT) reporter gene linked to 326 bp of the cytochrome c promoter. Cytochrome c mRNA in stimulated muscles increased by 1.3- to 1.7-fold above control between 1 and 7 days. Cytochrome c protein was increased after 5 days of stimulation to reach levels that were 1.9-fold higher than control by 7 days. Cytochrome c mRNA stability, determined with an in vitro decay assay, was greater in stimulated TA than in control between 2 and 4 days, likely mediated by the induction of a cytosolic factor. In contrast, cytochrome c transcriptional activation was elevated only after 5 days of stimulation when mRNA stability...
Physiological Reports, 2017
PloS one, 2017
Obesity is clearly associated with an increased risk of breast cancer in postmenopausal women. Th... more Obesity is clearly associated with an increased risk of breast cancer in postmenopausal women. The purpose was to determine if obesity alters the adipocyte adipokine secretion profile, thereby altering the adipose-dependent paracrine/endocrine growth microenvironment surrounding breast cancer cells (MCF7). Additionally, we determined whether resveratrol (RSV) supplementation can counteract any obesity-dependent effects on breast cancer tumor growth microenvironment. Obese ZDF rats received standard chow diet or diet supplemented with 200 mg/kg body weight RSV. Chow-fed Zucker rats served as lean controls. After 6 weeks, conditioned media (CM) prepared from inguinal subcutaneous adipose tissue (scAT) was added to MCF7 cells for 24 hrs. Experiments were also conducted using purified isolated adipocytes to determine whether any endocrine effects could be attributed specifically to the adipocyte component of adipose tissue. scAT from ZDF rats promoted cell cycle entry in MCF7 cells whic...
Biochemical Journal, 1995
Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, im... more Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, import and refolding of precursor proteins into mitochondria. The purpose of the present study was to evaluate the relationship between mitochondrial content under steady-state conditions, and during the induction of organelle biogenesis, with the expression of stress proteins and mitochondrial chaperonins. A comparison of steady-state levels of mitochondrial enzyme activity [cytochrome c oxidase (CYTOX)] with chaperonin levels [the heat-shock protein HSP60, the glucose-regulated protein GRP75 (mtHSP70)] in striated muscles possessing a wide range of oxidative capacities revealed a proportional expression between the two. This relationship was disrupted by chronic contractile activity brought about by 10 days of 10 Hz stimulation of the tibialis anterior (TA) muscle, which induced 2.4-fold increases in CYTOX activity, but 3.2- and 9.3-fold increases in HSP60 and GRP75 respectively. The ind...
American journal of physiology. Cell physiology, 2000
Mitochondrial biogenesis is accompanied by an increased expression of components of the protein i... more Mitochondrial biogenesis is accompanied by an increased expression of components of the protein import machinery, as well as increased import of proteins destined for the matrix. We evaluated the role of the outer membrane receptor Tom20 by varying its expression and measuring changes in the import of malate dehydrogenase (MDH) in differentiating C2C12 muscle cells. Cells transfected with Tom20 had levels that were twofold higher than in control cells. Labeling of cells followed by immunoprecipitation of MDH revealed equivalent increases in MDH import. This parallelism between import rate and Tom20 levels was also evident as a result of thyroid hormone treatment. Using antisense oligodeoxynucleotides, we inhibited Tom20 expression by 40%, resulting in 40-60% reductions in MDH import. In vitro assays also revealed that import into the matrix was more sensitive to Tom20 inhibition than import into the outer membrane. These data indicate a close relationship between induced changes in ...
Journal of applied physiology (Bethesda, Md. : 1985), 2000
To determine the role of intramitochondrial protein synthesis (PS) and degradation (PD) in contra... more To determine the role of intramitochondrial protein synthesis (PS) and degradation (PD) in contractile activity-induced mitochondrial biogenesis, we evaluated rates of [(35)S]methionine incorporation into protein in isolated rat muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Rates of PS ranged from 47 to 125% greater (P < 0.05) in IMF compared with SS mitochondria. Intense, acute in situ contractile activity (10 Hz, 5 min) of fast-twitch gastrocnemius muscle resulted in a 50% decrease in PS (P < 0.05) in SS but not IMF mitochondria. Recovery, or continued contractile activity (55 min), reestablished PS in SS mitochondria. In contrast, PS was not affected in either SS or IMF mitochondria after prolonged (60-min) contractile activity in the presence or absence of a recovery period. PD was not influenced by 5 min of contractile activity in the presence or absence of recovery but was reduced after 60 min of contractions followed by recovery. Chronic stimulati...
Molecular Cancer Research, 2005
Ring finger proteins serve many vital functions within the cell. We have identified RNF11, a nove... more Ring finger proteins serve many vital functions within the cell. We have identified RNF11, a novel 154-amino acid ring finger-containing protein, which is elevated in breast cancer. Within its ring finger domain, RNF11 contains an AKT phosphorylation site (T135) that is situated within a 14-3-3 binding domain. In WM239 cells with constitutively active AKT, RNF11 exhibits seven distinct phosphopeptides as measured using two-dimensional phosphopeptide mapping. Upon inhibition of the AKT pathway or mutation of T135, the phosphorylation at one of these sites is virtually eliminated, suggesting that AKT may phosphorylate RNF11 at T135. Moreover, RNF11 is phosphorylated by AKT in vitro and is recognized by phospho-AKT substrate antibodies. RNF11 shows enhanced binding to 14-3-3 in WM239 cells compared with that seen in the parental WM35 cells which have low AKT activity. Furthermore, treatment of WM239 cells with LY294002 reduces RNF11/14-3-3 interactions suggesting that RNF11/14-3-3 binding is regulated by AKT. In addition, RNF11/14-3-3 binding is enhanced by constitutively active AKT and is diminished by dominant-negative AKT. There is also reduced 14-3-3 binding to T135E RNF11. RNF11 localization was altered from the cytoplasm to the nucleus by activated AKT. Thus, phosphorylation of RNF11 by AKT either causes its nuclear localization or induces degradation of cytoplasmic RNF11. In addition, T135E RNF11, which does not bind 14-3-3 and is not phosphorylated by AKT, causes a greater enhancement of transforming growth factor-B signaling than wild-type RNF11. It is clear that RNF11 function, localization, and potentially, degradation are regulated by AKT. Disregulation of proper RNF11 function by AKT may prove to be detrimental to patient outcomes, making RNF11 a potential target for novel cancer therapeutics.
Journal of Proteome Research, 2005
Protein expression becomes altered in breast epithelium during malignant transformation. Knowledg... more Protein expression becomes altered in breast epithelium during malignant transformation. Knowledge of these perturbations should provide insight into the molecular basis of breast cancer, as well as reveal possible new therapeutic targets. To this end, we have performed an extensive comparative proteomic survey of global protein expression patterns in proliferating MCF-7 breast cancer cells and normal human mammary epithelial cells using gel-free shotgun tandem mass spectrometry. Pathophysiological alterations associated with the malignant breast cancer phenotype were detected, including differences in the apparent levels of key regulators of the cell cycle, signal transduction, apoptosis, transcriptional regulation, and cell metabolism.
Journal of Biological Chemistry, 2001
Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mito... more Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochondrial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (؊726 base pairs) or minimal (؊66 base pairs) cytochrome c promoter/chloramphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the ؊726 promoter was abolished when muscle contraction was inhibited at various subcellular locations by pretreatment with either the Na ؉ channel blocker tetrodotoxin, the intracellular Ca 2؉ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochondrial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter, electromobility shift assays performed within the first intron (؉75 to ؉104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.
Archives of Biochemistry and Biophysics, 1996
both nuclear-and mitochondrially encoded mRNAs. Steady-state levels of mRNAs encoding mitochon-We... more both nuclear-and mitochondrially encoded mRNAs. Steady-state levels of mRNAs encoding mitochon-We have previously shown that the mRNAs encoding drial proteins are drastically different among tissues. subunit VIc (nuclear-encoded) and subunit III (mito-We evaluated tissue-specific variations in mRNA stachondrially encoded) are coordinately expressed in varbility by comparing rates of mRNA decay in liver, ious tissues of the rat possessing a wide range of mitoheart, and muscle following the inhibition of tranchondrial contents (1). A coordinated expression of scription. Rates of decline of the mRNAs encoding dthese mRNAs may be important for the assembly of aminolevulinate synthase (ALAs), cytochrome c oxiholoenzyme subunits in a 1:1 stoichiometry (2). Steadydase subunit VIc (nuclear-encoded), and subunit III state mRNA levels result from a balance between the (mitochondrially encoded) in heart, liver, and muscle rates of mRNA transcription and mRNA degradation. for 6 h following transcription inhibition with actino-Modifications of the mRNA transcript with respect mycin D or ethidium bromide were measured. Subunit to secondary structure, polyadenylation state, or its VIc mRNA levels were least stable in liver (t 1/2 Å 2.4 h), interaction with protein transacting factors are now slightly greater in heart (t 1/2 Å 3.3 h), and very stable recognized as important mechanisms through which in skeletal muscle. Similarly, ALAs mRNA exhibited a changes in mRNA stability could have profound effects t 1/2 of 41 min in liver, but this was markedly increased on tissue mRNA levels (3-5). To date, limited informato approximately 11-14 h in heart and skeletal muscle. tion is available on the stability of nuclear-derived (6, 7) In contrast, subunit III was least stable in heart (t 1/2 Å and mitochondrially derived (8-10) mRNAs encoding 2.1 h), somewhat more stable in liver (t 1/2 Å 3.8 h), but proteins destined for the mitochondrion, particularly no decline in subunit III mRNA levels occurred in musrelated to tissue specificity. Large variations in tissuecle following the inhibition of transcription. Thus, specific rates of mRNA degradation could account for muscle, heart, and liver possess tissue-specific mechathe difference in steady-state mRNA levels observed nisms which control the stability of mRNAs encoding mitochondrial proteins. In addition, the coordinated between tissues. This study was undertaken to deterexpression of subunit III and VIc mRNAs in different mine if parallelism in the rates of CYTOX mRNA degtissues is partly due to parallel rates of mRNA turnradation could account for the coordinated expression over. This suggests the presence of intra-and extramiof these mRNAs in different tissues. In addition, since tochondrial factors within a tissue which regulate the heme is an important functional component of the CYstability of specific mRNAs in a similar manner. ᭧ 1996 TOX holoenzyme, an additional purpose was to evalu-Academic Press, Inc. ate the mRNA stability of the nuclear-encoded enzyme Key Words: cytochrome c oxidase, gene expression, d-aminolevulinate synthase (ALAs), the rate-limiting heme metabolism, mitochondrial biogenesis, mRNA enzyme in heme biosynthesis (11). The half-life (t 1/2) of stability. ALAs mRNA has been reported to be 20 min in liver (12) and we wished to compare this t 1/2 value in liver Cytochrome c oxidase (CYTOX) 2 is a multisubunit with that found in muscle and heart to provide some enzyme consisting of proteins that are translated from insight into the possibility of tissue-specific regulation of mRNA stability.
AJP: Cell Physiology, 2003
Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca2... more Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca2+ in muscle cells. To characterize the potential role of Ca2+ as a messenger involved in mitochondrial biogenesis in muscle, we determined the effects of the Ca2+ ionophore A-23187 on the expression of nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 μM A-23187 for 48–96 h increased nuclear-encoded β-subunit F1ATPase and malate dehydrogenase (MDH) mRNA levels by 50–100% ( P…
Journal of applied physiology (Bethesda, Md. : 1985), Jan 5, 2016
The molecular mechanisms behind the obesity-breast cancer association may be regulated via adipok... more The molecular mechanisms behind the obesity-breast cancer association may be regulated via adipokine secretion by white adipose tissue. Specifically, adiponectin (ADIPO) and leptin (LEP) are altered with adiposity and exert antagonistic effects on cancer cell proliferation. We set out to determine whether altering adiposity in-vivo via high fat diet (HFD) feeding changed the tumor growth supporting nature of adipose tissue and if voluntary physical activity (VPA) could ameliorate these HFD-dependent effects. We show that conditioned media (CM) created from the adipose tissue of HFD fed animals caused an increase in the proliferation of MCF7 cells compared to cells exposed to CM prepared from the adipose of lean chow diet fed counterparts. This increased proliferation was driven within the MCF7 cells by an HFD-dependent antagonism between AMPK and Akt signaling pathways, decreasing p27 protein levels via reduced phosphorylation at T198 and downregulation of AdiporR1. VPA can ameliora...
10 other HighWire hosted articles, the first 5 are: This article has been cited by [PDF] [Full Te... more 10 other HighWire hosted articles, the first 5 are: This article has been cited by [PDF] [Full Text] [Abstract] , June 15, 2006; 209 (12): 2265-2275. J Exp Biol Coordination of metabolic plasticity in skeletal muscle [PDF] [Full Text] [Abstract] , April 1, 2007; 292 (4): H1764-H1769. Am J Physiol Heart Circ Physiol reduced gap junctional and mitochondrial levels of connexin 43 Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with [PDF] [Full Text] [Abstract] , September 1, 2007; 293 (3): E672-E680. Am J Physiol Endocrinol Metab proteins in skeletal muscle of patients with mtDNA defects The effect of training on the expression of mitochondrial biogenesis-and apoptosis-related [PDF] [Full Text] [Abstract] , January 1, 2008; 294 (1): H249-H256. Am J Physiol Heart Circ Physiol protein 70 overexpression Mitochondria protection from hypoxia/reoxygenation injury with mitochondria heat shock [PDF] [Full Text] [Abstract] , January , 2011; 300 (1): C138-C145. Am J Physiol Cell Physiol Effect of denervation-induced muscle disuse on mitochondrial protein import including high resolution figures, can be found at: Updated information and services
Theriau CF, Shpilberg Y, Riddell MC, Connor MK. Voluntary physical activity abolishes the prolife... more Theriau CF, Shpilberg Y, Riddell MC, Connor MK. Voluntary physical activity abolishes the proliferative tumor growth microenvironment created by adipose tissue in animals fed a high fat diet.The molecular mechanisms behind the obesity-breast cancer association may be regulated via adipokine secretion by white adipose tissue. Specifically , adiponectin and leptin are altered with adiposity and exert antagonistic effects on cancer cell proliferation. We set out to determine whether altering adiposity in vivo via high fat diet (HFD) feeding changed the tumor growth supporting nature of adipose tissue and whether voluntary physical activity (PA) could ameliorate these HFD-dependent effects. We show that conditioned media (CM) created from the adipose tissue of HFD fed animals caused an increase in the proliferation of MCF7 cells compared with cells exposed to CM prepared from the adipose of lean chow diet fed counterparts. This increased proliferation was driven within the MCF7 cells by an HFD-dependent antagonism between AMP-activated protein kinase (AMPK) and protein kinase B (Akt) signaling pathways, decreasing p27 protein levels via reduced phosphorylation at T198 and down-regulation of adiponectin receptor 1 (AdipoR1). PA can ameliorate these proliferative effects of HFD-CM on MCF7 cells, increasing p27 T198 by AMPK, reducing pAkt T308 , and increasing AdipoR1, resulting in cell cycle withdrawal in a manner that depends on the PA intensity. High physical activity (Ͼ3 km/day) completely abolished the effects of HFD feeding. In addition, AdipoR1 overexpression mimics the effects of exercise, abolishing the proliferative effects of the HFD-CM on MCF7 cells and further enhancing the antiprolifera-tive effects of PA on the HFD-CM. Thus voluntary PA represents a means to counteract the proliferative effects of adipose tissue on breast cancers in obese patients. physical activity; obesity; adipokines; breast cancer NEW & NOTEWORTHY We hypothesized that voluntary physical activity (PA) would counteract the deleterious adipose-dependent growth microen-vironment to which a breast cancer is exposed. We show that PA altered the adipokine secretion profile of adipose in a volume-dependent manner. This alteration resulted in growth inhibition of estrogen receptor positive breast cancer cells in culture. Furthermore, stabilizing adiponectin receptor 1 expression in the cancer cells made them resistant to the cell cycle entry effects that accompany obesity.
both nuclear-and mitochondrially encoded mRNAs. Steady-state levels of mRNAs encoding mitochon-We... more both nuclear-and mitochondrially encoded mRNAs. Steady-state levels of mRNAs encoding mitochon-We have previously shown that the mRNAs encoding drial proteins are drastically different among tissues. subunit VIc (nuclear-encoded) and subunit III (mito-We evaluated tissue-specific variations in mRNA sta-chondrially encoded) are coordinately expressed in var-bility by comparing rates of mRNA decay in liver, ious tissues of the rat possessing a wide range of mito-heart, and muscle following the inhibition of tran-chondrial contents (1). A coordinated expression of scription. Rates of decline of the mRNAs encoding d-these mRNAs may be important for the assembly of aminolevulinate synthase (ALAs), cytochrome c oxi-holoenzyme subunits in a 1:1 stoichiometry (2). Steady-dase subunit VIc (nuclear-encoded), and subunit III state mRNA levels result from a balance between the (mitochondrially encoded) in heart, liver, and muscle rates of mRNA transcription and mRNA degradation. for 6 h following transcription inhibition with actino-Modifications of the mRNA transcript with respect mycin D or ethidium bromide were measured. Subunit to secondary structure, polyadenylation state, or its VIc mRNA levels were least stable in liver (t 1/2 Å 2.4 h), interaction with protein transacting factors are now slightly greater in heart (t 1/2 Å 3.3 h), and very stable recognized as important mechanisms through which in skeletal muscle. Similarly, ALAs mRNA exhibited a changes in mRNA stability could have profound effects t 1/2 of 41 min in liver, but this was markedly increased on tissue mRNA levels (3-5). To date, limited informa-to approximately 11-14 h in heart and skeletal muscle. tion is available on the stability of nuclear-derived (6, 7) In contrast, subunit III was least stable in heart (t 1/2 Å and mitochondrially derived (8-10) mRNAs encoding 2.1 h), somewhat more stable in liver (t 1/2 Å 3.8 h), but proteins destined for the mitochondrion, particularly no decline in subunit III mRNA levels occurred in mus-related to tissue specificity. Large variations in tissue-cle following the inhibition of transcription. Thus, specific rates of mRNA degradation could account for muscle, heart, and liver possess tissue-specific mecha-the difference in steady-state mRNA levels observed nisms which control the stability of mRNAs encoding mitochondrial proteins. In addition, the coordinated between tissues. This study was undertaken to deter-expression of subunit III and VIc mRNAs in different mine if parallelism in the rates of CYTOX mRNA deg-tissues is partly due to parallel rates of mRNA turn-radation could account for the coordinated expression over. This suggests the presence of intra-and extrami-of these mRNAs in different tissues. In addition, since tochondrial factors within a tissue which regulate the heme is an important functional component of the CY-stability of specific mRNAs in a similar manner. ᭧ 1996 TOX holoenzyme, an additional purpose was to evalu-Academic Press, Inc. ate the mRNA stability of the nuclear-encoded enzyme Key Words: cytochrome c oxidase, gene expression, d-aminolevulinate synthase (ALAs), the rate-limiting heme metabolism, mitochondrial biogenesis, mRNA enzyme in heme biosynthesis (11). The half-life (t 1/2) of stability.
Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mito... more Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochon-drial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (؊726 base pairs) or minimal (؊66 base pairs) cytochrome c promoter/chlor-amphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the ؊726 promoter was abolished when muscle contraction was inhibited at various subcellu-lar locations by pretreatment with either the Na ؉ channel blocker tetrodotoxin, the intracellular Ca 2؉ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tet-raacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochon-drial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter , electromobility shift assays performed within the first intron (؉75 to ؉104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.
Nature Medicine, 2002
Cell-cycle deregulation is a hallmark of cancer. Loss of cytokinemediated G1 arrest may confer an... more Cell-cycle deregulation is a hallmark of cancer. Loss of cytokinemediated G1 arrest may confer an advantage during malignant progression. Resistance to the antiproliferative effects of transforming growth factor-β (TGF-β) often occurs despite intact TGFβ signaling and such cells may manifest resistance to multiple inhibitory cytokines, suggesting underlying alterations in cellcycle controls 1,2. Cyclin-dependent kinases (cdks) are regulated by cyclin binding, phosphorylation and by two families of cdk inhibitors 3. G1 progression is governed by D-type and E-type cyclin-cdk complexes. The inhibitors of cdk4 (INK4) family includes p15 INK4B and the kinase inhibitor protein (KIP) family comprises p21 Cip1 , p27 Kip1 and p57 Kip2 (ref. 4). In addition to inhibition of cyclin E-cdk2, p21 and p27 also facilitate assembly and activation of cyclin D-cdks in early G1 (refs. 5,6). p27 Kip1 was first identified as a mediator of TGF-β1-induced G1 arrest 7-9. TGF-β induces expression of the gene encoding p15 INK4B. p15 INK4B binds and inhibits cdk4 facilitating dissociation of p27 and cyclin D1 from cyclin D1-cdk4-p27 complexes and p27 binds and inhibits cyclin E-cdk2, leading to G1 arrest 10-12. p27 also mediates G1 arrest induced by IL-6 (ref. 13). p27 is a nuclear protein whose frequent deregulation in human cancers may confer resistance to antiproliferative signals. In cMyc or MAPK activated cancer-derived lines, cyclin D1-cdk4/6 complexes sequester p27, and cyclin E-cdk2-inhibition is impaired 14-16. cMyc inhibits p15 induction by TGF-β (ref. 17) and may also induce a factor that inactivates p27 18. In up to 50% of human cancers, reduced p27 protein is associated with a poor prognosis 19. In some tumors, p27 is mislocalized to the cytoplasm 19,20 , however; the mechanism and significance of this has not been elucidated. In human cancers, constitutive activation of phosphoinositol 3′ kinase (PI3K) and its effector PKB/Akt arise through oncogenic receptor tyrosine kinase activation, Ras activation, mutational loss of PTEN, or through activating mutation of the PI3K effector, protein kinase B (PKB)/Akt (hereafter termed Akt) itself 21,22. Akt can increase cyclin D1 levels 23 and downregulate p27 by increasing p27 proteolysis 24 or repressing p27 expression through Akt phosphorylation of a forkhead transcription factor 25. However, in most cancers, reduced p27 does not result from transcriptional silencing 19. Here we show that Akt causes resistance to cytokine-mediated G1 arrest. p27 phosphorylation by Akt impairs its nuclear import and leads to cytoplasmic p27 accumulation. In human breast cancers, cytoplasmic mislocalization of p27 is associated with Akt activation, loss of differentiation and poor patient outcome. Activation of Akt in lines resistant to G1 arrest by TGF-β Ras has been shown to confer TGF-β resistance. While investigating mechanisms of TGF-β resistance, we found that two TGF-β PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest
The Journal of Urology, 2012
American Journal of Physiology-Endocrinology and Metabolism, 1999
We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation ... more We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation and mRNA stability with unilateral chronic stimulation (10 Hz, 3 h/day) of the rat tibialis anterior (TA) muscle for 1, 2, 3, 4, 5, and 7 days ( n = 3–11/group). Transcriptional activation was assessed by direct plasmid DNA injection into the TA with a chloramphenicol acetyltransferase (CAT) reporter gene linked to 326 bp of the cytochrome c promoter. Cytochrome c mRNA in stimulated muscles increased by 1.3- to 1.7-fold above control between 1 and 7 days. Cytochrome c protein was increased after 5 days of stimulation to reach levels that were 1.9-fold higher than control by 7 days. Cytochrome c mRNA stability, determined with an in vitro decay assay, was greater in stimulated TA than in control between 2 and 4 days, likely mediated by the induction of a cytosolic factor. In contrast, cytochrome c transcriptional activation was elevated only after 5 days of stimulation when mRNA stability...
Physiological Reports, 2017
PloS one, 2017
Obesity is clearly associated with an increased risk of breast cancer in postmenopausal women. Th... more Obesity is clearly associated with an increased risk of breast cancer in postmenopausal women. The purpose was to determine if obesity alters the adipocyte adipokine secretion profile, thereby altering the adipose-dependent paracrine/endocrine growth microenvironment surrounding breast cancer cells (MCF7). Additionally, we determined whether resveratrol (RSV) supplementation can counteract any obesity-dependent effects on breast cancer tumor growth microenvironment. Obese ZDF rats received standard chow diet or diet supplemented with 200 mg/kg body weight RSV. Chow-fed Zucker rats served as lean controls. After 6 weeks, conditioned media (CM) prepared from inguinal subcutaneous adipose tissue (scAT) was added to MCF7 cells for 24 hrs. Experiments were also conducted using purified isolated adipocytes to determine whether any endocrine effects could be attributed specifically to the adipocyte component of adipose tissue. scAT from ZDF rats promoted cell cycle entry in MCF7 cells whic...
Biochemical Journal, 1995
Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, im... more Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, import and refolding of precursor proteins into mitochondria. The purpose of the present study was to evaluate the relationship between mitochondrial content under steady-state conditions, and during the induction of organelle biogenesis, with the expression of stress proteins and mitochondrial chaperonins. A comparison of steady-state levels of mitochondrial enzyme activity [cytochrome c oxidase (CYTOX)] with chaperonin levels [the heat-shock protein HSP60, the glucose-regulated protein GRP75 (mtHSP70)] in striated muscles possessing a wide range of oxidative capacities revealed a proportional expression between the two. This relationship was disrupted by chronic contractile activity brought about by 10 days of 10 Hz stimulation of the tibialis anterior (TA) muscle, which induced 2.4-fold increases in CYTOX activity, but 3.2- and 9.3-fold increases in HSP60 and GRP75 respectively. The ind...
American journal of physiology. Cell physiology, 2000
Mitochondrial biogenesis is accompanied by an increased expression of components of the protein i... more Mitochondrial biogenesis is accompanied by an increased expression of components of the protein import machinery, as well as increased import of proteins destined for the matrix. We evaluated the role of the outer membrane receptor Tom20 by varying its expression and measuring changes in the import of malate dehydrogenase (MDH) in differentiating C2C12 muscle cells. Cells transfected with Tom20 had levels that were twofold higher than in control cells. Labeling of cells followed by immunoprecipitation of MDH revealed equivalent increases in MDH import. This parallelism between import rate and Tom20 levels was also evident as a result of thyroid hormone treatment. Using antisense oligodeoxynucleotides, we inhibited Tom20 expression by 40%, resulting in 40-60% reductions in MDH import. In vitro assays also revealed that import into the matrix was more sensitive to Tom20 inhibition than import into the outer membrane. These data indicate a close relationship between induced changes in ...
Journal of applied physiology (Bethesda, Md. : 1985), 2000
To determine the role of intramitochondrial protein synthesis (PS) and degradation (PD) in contra... more To determine the role of intramitochondrial protein synthesis (PS) and degradation (PD) in contractile activity-induced mitochondrial biogenesis, we evaluated rates of [(35)S]methionine incorporation into protein in isolated rat muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Rates of PS ranged from 47 to 125% greater (P < 0.05) in IMF compared with SS mitochondria. Intense, acute in situ contractile activity (10 Hz, 5 min) of fast-twitch gastrocnemius muscle resulted in a 50% decrease in PS (P < 0.05) in SS but not IMF mitochondria. Recovery, or continued contractile activity (55 min), reestablished PS in SS mitochondria. In contrast, PS was not affected in either SS or IMF mitochondria after prolonged (60-min) contractile activity in the presence or absence of a recovery period. PD was not influenced by 5 min of contractile activity in the presence or absence of recovery but was reduced after 60 min of contractions followed by recovery. Chronic stimulati...
Molecular Cancer Research, 2005
Ring finger proteins serve many vital functions within the cell. We have identified RNF11, a nove... more Ring finger proteins serve many vital functions within the cell. We have identified RNF11, a novel 154-amino acid ring finger-containing protein, which is elevated in breast cancer. Within its ring finger domain, RNF11 contains an AKT phosphorylation site (T135) that is situated within a 14-3-3 binding domain. In WM239 cells with constitutively active AKT, RNF11 exhibits seven distinct phosphopeptides as measured using two-dimensional phosphopeptide mapping. Upon inhibition of the AKT pathway or mutation of T135, the phosphorylation at one of these sites is virtually eliminated, suggesting that AKT may phosphorylate RNF11 at T135. Moreover, RNF11 is phosphorylated by AKT in vitro and is recognized by phospho-AKT substrate antibodies. RNF11 shows enhanced binding to 14-3-3 in WM239 cells compared with that seen in the parental WM35 cells which have low AKT activity. Furthermore, treatment of WM239 cells with LY294002 reduces RNF11/14-3-3 interactions suggesting that RNF11/14-3-3 binding is regulated by AKT. In addition, RNF11/14-3-3 binding is enhanced by constitutively active AKT and is diminished by dominant-negative AKT. There is also reduced 14-3-3 binding to T135E RNF11. RNF11 localization was altered from the cytoplasm to the nucleus by activated AKT. Thus, phosphorylation of RNF11 by AKT either causes its nuclear localization or induces degradation of cytoplasmic RNF11. In addition, T135E RNF11, which does not bind 14-3-3 and is not phosphorylated by AKT, causes a greater enhancement of transforming growth factor-B signaling than wild-type RNF11. It is clear that RNF11 function, localization, and potentially, degradation are regulated by AKT. Disregulation of proper RNF11 function by AKT may prove to be detrimental to patient outcomes, making RNF11 a potential target for novel cancer therapeutics.
Journal of Proteome Research, 2005
Protein expression becomes altered in breast epithelium during malignant transformation. Knowledg... more Protein expression becomes altered in breast epithelium during malignant transformation. Knowledge of these perturbations should provide insight into the molecular basis of breast cancer, as well as reveal possible new therapeutic targets. To this end, we have performed an extensive comparative proteomic survey of global protein expression patterns in proliferating MCF-7 breast cancer cells and normal human mammary epithelial cells using gel-free shotgun tandem mass spectrometry. Pathophysiological alterations associated with the malignant breast cancer phenotype were detected, including differences in the apparent levels of key regulators of the cell cycle, signal transduction, apoptosis, transcriptional regulation, and cell metabolism.
Journal of Biological Chemistry, 2001
Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mito... more Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochondrial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (؊726 base pairs) or minimal (؊66 base pairs) cytochrome c promoter/chloramphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the ؊726 promoter was abolished when muscle contraction was inhibited at various subcellular locations by pretreatment with either the Na ؉ channel blocker tetrodotoxin, the intracellular Ca 2؉ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochondrial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter, electromobility shift assays performed within the first intron (؉75 to ؉104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.
Archives of Biochemistry and Biophysics, 1996
both nuclear-and mitochondrially encoded mRNAs. Steady-state levels of mRNAs encoding mitochon-We... more both nuclear-and mitochondrially encoded mRNAs. Steady-state levels of mRNAs encoding mitochon-We have previously shown that the mRNAs encoding drial proteins are drastically different among tissues. subunit VIc (nuclear-encoded) and subunit III (mito-We evaluated tissue-specific variations in mRNA stachondrially encoded) are coordinately expressed in varbility by comparing rates of mRNA decay in liver, ious tissues of the rat possessing a wide range of mitoheart, and muscle following the inhibition of tranchondrial contents (1). A coordinated expression of scription. Rates of decline of the mRNAs encoding dthese mRNAs may be important for the assembly of aminolevulinate synthase (ALAs), cytochrome c oxiholoenzyme subunits in a 1:1 stoichiometry (2). Steadydase subunit VIc (nuclear-encoded), and subunit III state mRNA levels result from a balance between the (mitochondrially encoded) in heart, liver, and muscle rates of mRNA transcription and mRNA degradation. for 6 h following transcription inhibition with actino-Modifications of the mRNA transcript with respect mycin D or ethidium bromide were measured. Subunit to secondary structure, polyadenylation state, or its VIc mRNA levels were least stable in liver (t 1/2 Å 2.4 h), interaction with protein transacting factors are now slightly greater in heart (t 1/2 Å 3.3 h), and very stable recognized as important mechanisms through which in skeletal muscle. Similarly, ALAs mRNA exhibited a changes in mRNA stability could have profound effects t 1/2 of 41 min in liver, but this was markedly increased on tissue mRNA levels (3-5). To date, limited informato approximately 11-14 h in heart and skeletal muscle. tion is available on the stability of nuclear-derived (6, 7) In contrast, subunit III was least stable in heart (t 1/2 Å and mitochondrially derived (8-10) mRNAs encoding 2.1 h), somewhat more stable in liver (t 1/2 Å 3.8 h), but proteins destined for the mitochondrion, particularly no decline in subunit III mRNA levels occurred in musrelated to tissue specificity. Large variations in tissuecle following the inhibition of transcription. Thus, specific rates of mRNA degradation could account for muscle, heart, and liver possess tissue-specific mechathe difference in steady-state mRNA levels observed nisms which control the stability of mRNAs encoding mitochondrial proteins. In addition, the coordinated between tissues. This study was undertaken to deterexpression of subunit III and VIc mRNAs in different mine if parallelism in the rates of CYTOX mRNA degtissues is partly due to parallel rates of mRNA turnradation could account for the coordinated expression over. This suggests the presence of intra-and extramiof these mRNAs in different tissues. In addition, since tochondrial factors within a tissue which regulate the heme is an important functional component of the CYstability of specific mRNAs in a similar manner. ᭧ 1996 TOX holoenzyme, an additional purpose was to evalu-Academic Press, Inc. ate the mRNA stability of the nuclear-encoded enzyme Key Words: cytochrome c oxidase, gene expression, d-aminolevulinate synthase (ALAs), the rate-limiting heme metabolism, mitochondrial biogenesis, mRNA enzyme in heme biosynthesis (11). The half-life (t 1/2) of stability. ALAs mRNA has been reported to be 20 min in liver (12) and we wished to compare this t 1/2 value in liver Cytochrome c oxidase (CYTOX) 2 is a multisubunit with that found in muscle and heart to provide some enzyme consisting of proteins that are translated from insight into the possibility of tissue-specific regulation of mRNA stability.
AJP: Cell Physiology, 2003
Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca2... more Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca2+ in muscle cells. To characterize the potential role of Ca2+ as a messenger involved in mitochondrial biogenesis in muscle, we determined the effects of the Ca2+ ionophore A-23187 on the expression of nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 μM A-23187 for 48–96 h increased nuclear-encoded β-subunit F1ATPase and malate dehydrogenase (MDH) mRNA levels by 50–100% ( P…
Journal of applied physiology (Bethesda, Md. : 1985), Jan 5, 2016
The molecular mechanisms behind the obesity-breast cancer association may be regulated via adipok... more The molecular mechanisms behind the obesity-breast cancer association may be regulated via adipokine secretion by white adipose tissue. Specifically, adiponectin (ADIPO) and leptin (LEP) are altered with adiposity and exert antagonistic effects on cancer cell proliferation. We set out to determine whether altering adiposity in-vivo via high fat diet (HFD) feeding changed the tumor growth supporting nature of adipose tissue and if voluntary physical activity (VPA) could ameliorate these HFD-dependent effects. We show that conditioned media (CM) created from the adipose tissue of HFD fed animals caused an increase in the proliferation of MCF7 cells compared to cells exposed to CM prepared from the adipose of lean chow diet fed counterparts. This increased proliferation was driven within the MCF7 cells by an HFD-dependent antagonism between AMPK and Akt signaling pathways, decreasing p27 protein levels via reduced phosphorylation at T198 and downregulation of AdiporR1. VPA can ameliora...
10 other HighWire hosted articles, the first 5 are: This article has been cited by [PDF] [Full Te... more 10 other HighWire hosted articles, the first 5 are: This article has been cited by [PDF] [Full Text] [Abstract] , June 15, 2006; 209 (12): 2265-2275. J Exp Biol Coordination of metabolic plasticity in skeletal muscle [PDF] [Full Text] [Abstract] , April 1, 2007; 292 (4): H1764-H1769. Am J Physiol Heart Circ Physiol reduced gap junctional and mitochondrial levels of connexin 43 Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with [PDF] [Full Text] [Abstract] , September 1, 2007; 293 (3): E672-E680. Am J Physiol Endocrinol Metab proteins in skeletal muscle of patients with mtDNA defects The effect of training on the expression of mitochondrial biogenesis-and apoptosis-related [PDF] [Full Text] [Abstract] , January 1, 2008; 294 (1): H249-H256. Am J Physiol Heart Circ Physiol protein 70 overexpression Mitochondria protection from hypoxia/reoxygenation injury with mitochondria heat shock [PDF] [Full Text] [Abstract] , January , 2011; 300 (1): C138-C145. Am J Physiol Cell Physiol Effect of denervation-induced muscle disuse on mitochondrial protein import including high resolution figures, can be found at: Updated information and services
Theriau CF, Shpilberg Y, Riddell MC, Connor MK. Voluntary physical activity abolishes the prolife... more Theriau CF, Shpilberg Y, Riddell MC, Connor MK. Voluntary physical activity abolishes the proliferative tumor growth microenvironment created by adipose tissue in animals fed a high fat diet.The molecular mechanisms behind the obesity-breast cancer association may be regulated via adipokine secretion by white adipose tissue. Specifically , adiponectin and leptin are altered with adiposity and exert antagonistic effects on cancer cell proliferation. We set out to determine whether altering adiposity in vivo via high fat diet (HFD) feeding changed the tumor growth supporting nature of adipose tissue and whether voluntary physical activity (PA) could ameliorate these HFD-dependent effects. We show that conditioned media (CM) created from the adipose tissue of HFD fed animals caused an increase in the proliferation of MCF7 cells compared with cells exposed to CM prepared from the adipose of lean chow diet fed counterparts. This increased proliferation was driven within the MCF7 cells by an HFD-dependent antagonism between AMP-activated protein kinase (AMPK) and protein kinase B (Akt) signaling pathways, decreasing p27 protein levels via reduced phosphorylation at T198 and down-regulation of adiponectin receptor 1 (AdipoR1). PA can ameliorate these proliferative effects of HFD-CM on MCF7 cells, increasing p27 T198 by AMPK, reducing pAkt T308 , and increasing AdipoR1, resulting in cell cycle withdrawal in a manner that depends on the PA intensity. High physical activity (Ͼ3 km/day) completely abolished the effects of HFD feeding. In addition, AdipoR1 overexpression mimics the effects of exercise, abolishing the proliferative effects of the HFD-CM on MCF7 cells and further enhancing the antiprolifera-tive effects of PA on the HFD-CM. Thus voluntary PA represents a means to counteract the proliferative effects of adipose tissue on breast cancers in obese patients. physical activity; obesity; adipokines; breast cancer NEW & NOTEWORTHY We hypothesized that voluntary physical activity (PA) would counteract the deleterious adipose-dependent growth microen-vironment to which a breast cancer is exposed. We show that PA altered the adipokine secretion profile of adipose in a volume-dependent manner. This alteration resulted in growth inhibition of estrogen receptor positive breast cancer cells in culture. Furthermore, stabilizing adiponectin receptor 1 expression in the cancer cells made them resistant to the cell cycle entry effects that accompany obesity.
both nuclear-and mitochondrially encoded mRNAs. Steady-state levels of mRNAs encoding mitochon-We... more both nuclear-and mitochondrially encoded mRNAs. Steady-state levels of mRNAs encoding mitochon-We have previously shown that the mRNAs encoding drial proteins are drastically different among tissues. subunit VIc (nuclear-encoded) and subunit III (mito-We evaluated tissue-specific variations in mRNA sta-chondrially encoded) are coordinately expressed in var-bility by comparing rates of mRNA decay in liver, ious tissues of the rat possessing a wide range of mito-heart, and muscle following the inhibition of tran-chondrial contents (1). A coordinated expression of scription. Rates of decline of the mRNAs encoding d-these mRNAs may be important for the assembly of aminolevulinate synthase (ALAs), cytochrome c oxi-holoenzyme subunits in a 1:1 stoichiometry (2). Steady-dase subunit VIc (nuclear-encoded), and subunit III state mRNA levels result from a balance between the (mitochondrially encoded) in heart, liver, and muscle rates of mRNA transcription and mRNA degradation. for 6 h following transcription inhibition with actino-Modifications of the mRNA transcript with respect mycin D or ethidium bromide were measured. Subunit to secondary structure, polyadenylation state, or its VIc mRNA levels were least stable in liver (t 1/2 Å 2.4 h), interaction with protein transacting factors are now slightly greater in heart (t 1/2 Å 3.3 h), and very stable recognized as important mechanisms through which in skeletal muscle. Similarly, ALAs mRNA exhibited a changes in mRNA stability could have profound effects t 1/2 of 41 min in liver, but this was markedly increased on tissue mRNA levels (3-5). To date, limited informa-to approximately 11-14 h in heart and skeletal muscle. tion is available on the stability of nuclear-derived (6, 7) In contrast, subunit III was least stable in heart (t 1/2 Å and mitochondrially derived (8-10) mRNAs encoding 2.1 h), somewhat more stable in liver (t 1/2 Å 3.8 h), but proteins destined for the mitochondrion, particularly no decline in subunit III mRNA levels occurred in mus-related to tissue specificity. Large variations in tissue-cle following the inhibition of transcription. Thus, specific rates of mRNA degradation could account for muscle, heart, and liver possess tissue-specific mecha-the difference in steady-state mRNA levels observed nisms which control the stability of mRNAs encoding mitochondrial proteins. In addition, the coordinated between tissues. This study was undertaken to deter-expression of subunit III and VIc mRNAs in different mine if parallelism in the rates of CYTOX mRNA deg-tissues is partly due to parallel rates of mRNA turn-radation could account for the coordinated expression over. This suggests the presence of intra-and extrami-of these mRNAs in different tissues. In addition, since tochondrial factors within a tissue which regulate the heme is an important functional component of the CY-stability of specific mRNAs in a similar manner. ᭧ 1996 TOX holoenzyme, an additional purpose was to evalu-Academic Press, Inc. ate the mRNA stability of the nuclear-encoded enzyme Key Words: cytochrome c oxidase, gene expression, d-aminolevulinate synthase (ALAs), the rate-limiting heme metabolism, mitochondrial biogenesis, mRNA enzyme in heme biosynthesis (11). The half-life (t 1/2) of stability.
Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mito... more Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochon-drial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (؊726 base pairs) or minimal (؊66 base pairs) cytochrome c promoter/chlor-amphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the ؊726 promoter was abolished when muscle contraction was inhibited at various subcellu-lar locations by pretreatment with either the Na ؉ channel blocker tetrodotoxin, the intracellular Ca 2؉ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tet-raacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochon-drial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter , electromobility shift assays performed within the first intron (؉75 to ؉104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.