Zhonglin Chai - Academia.edu (original) (raw)

Papers by Zhonglin Chai

Journal of Biological Chemistry

The dynamin family of GTP-binding proteins has been implicated as playing an important role in en... more The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. In Drosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein betagamma subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the betagamma subunit are mediated by the pleckstrin homology domain of the GTPase...

Obesity research & clinical practice, 2007

To examine the relationship between family history of coronary heart disease (CHD), hypertension,... more To examine the relationship between family history of coronary heart disease (CHD), hypertension, and diabetes with risk of non-alcoholic fatty liver disease (NAFLD), and the possible interaction between these family histories and metabolic components for NAFLD. The 202 health office workers with no evidence of excessive alcohol drinking or hepatitis B or C virus infection were enrolled in the present study performed from March to June 2004. NAFLD was identified in 68 subjects by abdominal ultrasound. Logistic regression analysis showed that the presence of CHD family history increased the risk of NAFLD by 2.25-fold, 95% CI 1.1-4.1 (P = 0.014), while family history of diabetes or hypertension did not increase the risk. In combination with the presence of a family history of CHD, the effect on odds ratios (ORs) was increased for several metabolic features in predicting the incidence of NAFLD, including increased waist circumference, hypertriglyceridemia, hypertension and the occurren...

Journal of the American Society of Nephrology : JASN, 2013

Cell division autoantigen 1 (CDA1) enhances TGF-β signaling in renal and vascular cells, and rena... more Cell division autoantigen 1 (CDA1) enhances TGF-β signaling in renal and vascular cells, and renal expression of CDA1 is elevated in animal models of diabetes. In this study, we investigated the genetic deletion of Tspyl2, the gene encoding CDA1, in C57BL6 and ApoE knockout mice. The increased renal expression of TGF-β1, TGF-β type I and II receptors, and phosphorylated Smad3 associated with diabetes in wild-type mice was attenuated in diabetic CDA1 knockout mice. Notably, CDA1 deletion significantly reduced diabetes-associated renal matrix accumulation and immunohistochemical staining for collagens III and IV and attenuated glomerular and tubulointerstitial injury indices, despite the presence of persistent hyperglycemia, polyuria, renal hypertrophy, and hyperfiltration. Furthermore, CDA1 deletion reduced gene expression of TGF-β1 receptors in the kidney, resulting in a functionally attenuated response to exogenous TGF-β, including reduced levels of phosphorylated Smad3 and ERK1/2,...

Endocrinology, 2003

We searched expressed sequence tag databases with conserved domains of the short-chain alcohol de... more We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17 beta-hydroxysteroid dehydrogenase, 17 beta HSDXI. This enzyme converts 5 alpha-androstane-3 alpha, 17 beta-diol to androsterone. The substrate has been implicated in supporting gestation and modulating gamma-aminobutyric acid receptor activity. 17 beta HSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17 beta HSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5' untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17 beta HSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 ...

Genes, 2010

Cell Division Autoantigen 1 (CDA1) was discovered following screening a human expression library ... more Cell Division Autoantigen 1 (CDA1) was discovered following screening a human expression library with serum from a patient with Discoid Lupus Erythematosus. CDA1, encoded by TSPYL2 on the X chromosome, shares anti-proliferative and pro-fibrotic properties with TGF- It inhibits cell growth through p53, pERK1/2 and p21-mediated pathways and is implicated in tumorigenesis and the DNA damage response. Its pro-fibrotic property is mediated through cross-talk with TGF- that results in upregulation of extracellular matrix proteins. The latter properties have identified a key role for CDA1 in diabetes associated atherosclerosis. These dual properties place CDA1 as an attractive molecular target for treating tumors and vascular fibrosis including atherosclerosis and other vascular disorders associated with enhanced TGF-β action and tissue scarring.

PLoS ONE, 2013

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in... more Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced proinflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-b (TGF-b) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-b-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation. Citation: Tan SM, Sharma A, Yuen DYC, Stefanovic N, Krippner G, et al. (2013) The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice. PLoS ONE 8(7): e69193.

Journal of Biological Chemistry, 2001

We used an autoimmune serum from a patient with discoid lupus erythematosus to clone a cDNA of 28... more We used an autoimmune serum from a patient with discoid lupus erythematosus to clone a cDNA of 2808 base pairs. Its open reading frame of 2079 base pairs encodes a predicted polypeptide of 693 amino acids named CDA1 (cell division autoantigen-1). CDA1 has a predicted molecular mass of 79,430 Daltons and a pI of 4.26. The size of the cDNA is consistent with its estimated mRNA size. CDA1 comprises an N-terminal proline-rich domain, a central basic domain, and a C-terminal bipartite acidic domain. It has four putative nuclear localization signals and potential sites for phosphorylation by cAMP and cGMP-dependent kinases, protein kinase C, thymidine kinase, casein kinase II, and cyclin-dependent kinases (CDKs). CDA1 is phosphorylated in HeLa cells and by cyclin D1/CDK4, cyclin A/CDK2, and cyclin B/CDK1 in vitro. Its basic and acidic domains contain regions homologous to almost the entire human leukemia-associated SET protein. The same basic region is also homologous to nucleosome assembly proteins, testis TSPY protein, and an uncharacterized brain protein. CDA1 is present in the nuclear fraction of HeLa cells and localizes to the nucleus and nucleolus in HeLa cells transfected with CDA1 or its N terminus containing all four nuclear localization signals. Its acidic C terminus localizes mainly to the cytoplasm. CDA1 levels are low in serumstarved cells, increasing dramatically with serum stimulation. Expression of the CDA1 transgene, but not its N terminus, arrests HeLa cell growth, colony numbers, cell density, and bromodeoxyuridine uptake in a dose-dependent manner. The ability of CDA1 to arrest cell growth is abolished by mutation of the two CDK consensus phosphorylation sites. We propose that CDA1 is a negative regulator of cell growth and that its activity is regulated by its expression level and phosphorylation.

Journal of Biological Chemistry, 2007

We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells ar... more We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells arrests cell growth and inhibits DNA synthesis at S-phase (1). Here we show that CDA1induced arrest of cell growth is accompanied by increases in protein and mRNA levels of the cyclin-dependent kinase (Cdk) inhibitor protein, p21 Waf1/Cip1 (p21). Both p21 induction and cell growth arrest are reversed when CDA1 expression is inhibited. CDA1 also increases p53 protein, but not its mRNA, in a time-and dose-dependent manner. MDM2, a ubiquitin ligase regulating p53 degradation, is inactivated by CDA1, suggesting that p53 protein accumulation is due to decreased protein degradation. Knockdown of p53, using siRNA targeting two sites of p53 mRNA, abrogates transcriptional induction of p21 by CDA1. Deletion of the p53 responsive element in the distal region of p21 promoter attenuates promoter activity in response to CDA1. DNA damage caused by camptothecin treatment increases mRNA and protein levels of CDA1, accompanied by induction of p53. The DNA damage-induced p53 induction is markedly attenuated by CDA1 knockdown. CDA1 induces phosphorylation of ERK1/2(p44/42), an activity blocked by PD98059 and U0126, inhibitors of the upstream kinase MEK1/2. The MEK inhibitors also block induction of p21 mRNA and abrogate p21 promoter activity stimulated by CDA1. Cell cycle kinases, Cdk1, -2, -4, and -6 are inhibited by CDA1 overexpression. We conclude that CDA1 induces p53-and MEK/ERK1/2 MAPK-dependent expression of p21 by acting through the p53 responsive element in the p21 promoter and that this contributes to its antiproliferative activity.

European Journal of Cell Biology, 2000

Endocrinology, 2003

We searched expressed sequence tag databases with conserved domains of the short-chain alcohol de... more We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17 beta-hydroxysteroid dehydrogenase, 17 beta HSDXI. This enzyme converts 5 alpha-androstane-3 alpha, 17 beta-diol to androsterone. The substrate has been implicated in supporting gestation and modulating gamma-aminobutyric acid receptor activity. 17 beta HSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17 beta HSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5' untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17 beta HSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 or less adenosines (43% vs. 26%, P < 0.005). The C719T polymorphism is present in 15% of genomic DNA samples. Northern blot analysis showed high levels of 17 beta HSDXI expression in the pancreas, kidney, liver, lung, adrenal, ovary, and heart. Immunohistochemical staining for 17 beta HSDXI is strong in steroidogenic cells such as syncytiotrophoblasts, sebaceous gland, Leydig cells, and granulosa cells of the dominant follicle and corpus luteum. In the adrenal 17 beta HSDXI, staining colocalized with the distribution of 17 alpha-hydroxylase but was stronger in the mid to outer cortex. 17 beta HSDXI was also found in the fetus and increased after birth. Liver parenchymal cells and epithelium of the endometrium and small intestine also stained. Regulation studies in mouse Y1 cells showed that cAMP down-regulates 17 beta HSDXI enzymatic activity (40% vs. 32%, P < 0.05) and reduces gene expression to undetectable levels. All-trans-retinoic acid did not affect 17 beta HSDXI expression or activity, but addition of the retinoid together with cAMP significantly decreased activity over cAMP alone (32% vs. 23%, P < 0.05). Cloning and sequencing of the 17 beta HSDXI promoter identified the potential nuclear receptor steroidogenic factor-1 half-site TCCAAGGCCGG, and a cluster of three other potential steroidogenic factor-1 half-sites were found in the distal part of intron 1. Collectively, these results suggest a role for 17 beta HSDXI in androgen metabolism during steroidogenesis and a possible role in nonsteroidogenic tissues including paracrine modulation of 5 alpha-androstane-3 alpha, 17 beta-diol levels. 17 beta HSDXI could act by metabolizing compounds that stimulate steroid synthesis and/or by generating metabolites that inhibit it.

Diabetologia, 2010

Aims/hypothesis Excess accumulation of vascular extracellular matrix (ECM) is an important pathol... more Aims/hypothesis Excess accumulation of vascular extracellular matrix (ECM) is an important pathological process in cardiovascular diseases including diabetes-associated atherosclerosis. We explored how a recently identified molecule, cell division autoantigen 1 (CDA1), influences the profibrotic TGF-β pathway leading to vascular ECM accumulation. Methods Expression levels of genes encoding for CDA1, TGF-β and connective tissue growth factor (CTGF) were examined in aorta from Apoe −/− mice with or without diabetes. We used retroviral and adenoviral constructs to knockdown or overexpress Tspyl2, the gene encoding CDA1, in mouse vascular smooth muscle cells (VSMCs) with or without TGF-β treatment in order to demonstrate the role of CDA1 in TGF-β signalling. Results In vivo studies indicated that the mRNA levels of CDA1-encoding gene Tspyl2 and protein levels of CDA1 were elevated in the aorta of diabetic Apoe −/− mice, accompanied by increased levels of Tgf-β (also known as Tgfb1), Ctgf and ECM accumulation. In vitro studies in vascular cells showed that TGF-β treatment rapidly increased CDA1 protein levels, which then amplified TGF-β signalling leading to upregulation of ECM genes. Knockdown of CDA1-encoding gene Tspyl2 to reduce cellular CDA1 level markedly attenuated TGF-β-stimulated MAD homologue 3 (drosophila; SMAD3) phosphorylation and transcriptional activities. CDA1 overproduction increased and Tspyl2 knockdown decreased expression of TGF-β receptor type I, TβrI (also known as Tgfbr1), but not TGFβ receptor type II, TβrII (also known as Tgfbr2), providing a mechanism for CDA1's action in modulating TGF-β signalling. Knockdown of CDA1-encoding gene Tspyl2 also blocked the profibrotic effect of TGF-β in VSMCs. Conclusions/interpretation CDA1 plays an important role in vascular ECM accumulation by amplifying TGF-β signalling. This is critical for the profibrotic effect of TGF-β in the vasculature. CDA1 is therefore a potential target for attenuating vascular ECM accumulation caused by enhanced TGF-β action, as seen in diabetic atherosclerosis.

The American Journal of Cardiology, 2008

Kidney International, 2011

Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-b ... more Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-b (TGF-b) signaling in a number of cellular systems; here we found that its levels were elevated in the kidneys of two animal models of diabetic renal disease. The localization of CDA1 to tubular cells and podocytes in human kidney sections was similar to that seen in the rodent models. CDA1 small interfering RNA knockdown markedly attenuated, whereas its overexpression increased TGF-b signaling, modulating the expression of TGF-b, TGF-b receptors, connective tissue growth factor, collagen types I, III, IV, and fibronectin genes in HK-2 cells. CDA1 and TGF-b together were synergistic in stimulating TGF-b signaling and target gene expression. CDA1 knockdown effectively blocked TGF-b-stimulated expression of collagen genes. This was due to its ability to modulate the TGF-b type I, but not the type II, receptor, leading to increased phosphorylation of Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinase. Furthermore, the Smad3 inhibitor, SIS3, markedly attenuated the activities of CDA1 in stimulating TGF-b signaling as well as gene expression of collagens I, III, and IV. Thus, our in vitro and in vivo findings show that CDA1 has a critical role in TGF-b signaling in the kidney.

Journal of Biological Chemistry

The dynamin family of GTP-binding proteins has been implicated as playing an important role in en... more The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. In Drosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein betagamma subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the betagamma subunit are mediated by the pleckstrin homology domain of the GTPase...

Obesity research & clinical practice, 2007

To examine the relationship between family history of coronary heart disease (CHD), hypertension,... more To examine the relationship between family history of coronary heart disease (CHD), hypertension, and diabetes with risk of non-alcoholic fatty liver disease (NAFLD), and the possible interaction between these family histories and metabolic components for NAFLD. The 202 health office workers with no evidence of excessive alcohol drinking or hepatitis B or C virus infection were enrolled in the present study performed from March to June 2004. NAFLD was identified in 68 subjects by abdominal ultrasound. Logistic regression analysis showed that the presence of CHD family history increased the risk of NAFLD by 2.25-fold, 95% CI 1.1-4.1 (P = 0.014), while family history of diabetes or hypertension did not increase the risk. In combination with the presence of a family history of CHD, the effect on odds ratios (ORs) was increased for several metabolic features in predicting the incidence of NAFLD, including increased waist circumference, hypertriglyceridemia, hypertension and the occurren...

Journal of the American Society of Nephrology : JASN, 2013

Cell division autoantigen 1 (CDA1) enhances TGF-β signaling in renal and vascular cells, and rena... more Cell division autoantigen 1 (CDA1) enhances TGF-β signaling in renal and vascular cells, and renal expression of CDA1 is elevated in animal models of diabetes. In this study, we investigated the genetic deletion of Tspyl2, the gene encoding CDA1, in C57BL6 and ApoE knockout mice. The increased renal expression of TGF-β1, TGF-β type I and II receptors, and phosphorylated Smad3 associated with diabetes in wild-type mice was attenuated in diabetic CDA1 knockout mice. Notably, CDA1 deletion significantly reduced diabetes-associated renal matrix accumulation and immunohistochemical staining for collagens III and IV and attenuated glomerular and tubulointerstitial injury indices, despite the presence of persistent hyperglycemia, polyuria, renal hypertrophy, and hyperfiltration. Furthermore, CDA1 deletion reduced gene expression of TGF-β1 receptors in the kidney, resulting in a functionally attenuated response to exogenous TGF-β, including reduced levels of phosphorylated Smad3 and ERK1/2,...

Endocrinology, 2003

We searched expressed sequence tag databases with conserved domains of the short-chain alcohol de... more We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17 beta-hydroxysteroid dehydrogenase, 17 beta HSDXI. This enzyme converts 5 alpha-androstane-3 alpha, 17 beta-diol to androsterone. The substrate has been implicated in supporting gestation and modulating gamma-aminobutyric acid receptor activity. 17 beta HSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17 beta HSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5' untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17 beta HSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 ...

Genes, 2010

Cell Division Autoantigen 1 (CDA1) was discovered following screening a human expression library ... more Cell Division Autoantigen 1 (CDA1) was discovered following screening a human expression library with serum from a patient with Discoid Lupus Erythematosus. CDA1, encoded by TSPYL2 on the X chromosome, shares anti-proliferative and pro-fibrotic properties with TGF- It inhibits cell growth through p53, pERK1/2 and p21-mediated pathways and is implicated in tumorigenesis and the DNA damage response. Its pro-fibrotic property is mediated through cross-talk with TGF- that results in upregulation of extracellular matrix proteins. The latter properties have identified a key role for CDA1 in diabetes associated atherosclerosis. These dual properties place CDA1 as an attractive molecular target for treating tumors and vascular fibrosis including atherosclerosis and other vascular disorders associated with enhanced TGF-β action and tissue scarring.

PLoS ONE, 2013

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in... more Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced proinflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-b (TGF-b) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-b-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation. Citation: Tan SM, Sharma A, Yuen DYC, Stefanovic N, Krippner G, et al. (2013) The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice. PLoS ONE 8(7): e69193.

Journal of Biological Chemistry, 2001

We used an autoimmune serum from a patient with discoid lupus erythematosus to clone a cDNA of 28... more We used an autoimmune serum from a patient with discoid lupus erythematosus to clone a cDNA of 2808 base pairs. Its open reading frame of 2079 base pairs encodes a predicted polypeptide of 693 amino acids named CDA1 (cell division autoantigen-1). CDA1 has a predicted molecular mass of 79,430 Daltons and a pI of 4.26. The size of the cDNA is consistent with its estimated mRNA size. CDA1 comprises an N-terminal proline-rich domain, a central basic domain, and a C-terminal bipartite acidic domain. It has four putative nuclear localization signals and potential sites for phosphorylation by cAMP and cGMP-dependent kinases, protein kinase C, thymidine kinase, casein kinase II, and cyclin-dependent kinases (CDKs). CDA1 is phosphorylated in HeLa cells and by cyclin D1/CDK4, cyclin A/CDK2, and cyclin B/CDK1 in vitro. Its basic and acidic domains contain regions homologous to almost the entire human leukemia-associated SET protein. The same basic region is also homologous to nucleosome assembly proteins, testis TSPY protein, and an uncharacterized brain protein. CDA1 is present in the nuclear fraction of HeLa cells and localizes to the nucleus and nucleolus in HeLa cells transfected with CDA1 or its N terminus containing all four nuclear localization signals. Its acidic C terminus localizes mainly to the cytoplasm. CDA1 levels are low in serumstarved cells, increasing dramatically with serum stimulation. Expression of the CDA1 transgene, but not its N terminus, arrests HeLa cell growth, colony numbers, cell density, and bromodeoxyuridine uptake in a dose-dependent manner. The ability of CDA1 to arrest cell growth is abolished by mutation of the two CDK consensus phosphorylation sites. We propose that CDA1 is a negative regulator of cell growth and that its activity is regulated by its expression level and phosphorylation.

Journal of Biological Chemistry, 2007

We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells ar... more We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells arrests cell growth and inhibits DNA synthesis at S-phase (1). Here we show that CDA1induced arrest of cell growth is accompanied by increases in protein and mRNA levels of the cyclin-dependent kinase (Cdk) inhibitor protein, p21 Waf1/Cip1 (p21). Both p21 induction and cell growth arrest are reversed when CDA1 expression is inhibited. CDA1 also increases p53 protein, but not its mRNA, in a time-and dose-dependent manner. MDM2, a ubiquitin ligase regulating p53 degradation, is inactivated by CDA1, suggesting that p53 protein accumulation is due to decreased protein degradation. Knockdown of p53, using siRNA targeting two sites of p53 mRNA, abrogates transcriptional induction of p21 by CDA1. Deletion of the p53 responsive element in the distal region of p21 promoter attenuates promoter activity in response to CDA1. DNA damage caused by camptothecin treatment increases mRNA and protein levels of CDA1, accompanied by induction of p53. The DNA damage-induced p53 induction is markedly attenuated by CDA1 knockdown. CDA1 induces phosphorylation of ERK1/2(p44/42), an activity blocked by PD98059 and U0126, inhibitors of the upstream kinase MEK1/2. The MEK inhibitors also block induction of p21 mRNA and abrogate p21 promoter activity stimulated by CDA1. Cell cycle kinases, Cdk1, -2, -4, and -6 are inhibited by CDA1 overexpression. We conclude that CDA1 induces p53-and MEK/ERK1/2 MAPK-dependent expression of p21 by acting through the p53 responsive element in the p21 promoter and that this contributes to its antiproliferative activity.

European Journal of Cell Biology, 2000

Endocrinology, 2003

We searched expressed sequence tag databases with conserved domains of the short-chain alcohol de... more We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17 beta-hydroxysteroid dehydrogenase, 17 beta HSDXI. This enzyme converts 5 alpha-androstane-3 alpha, 17 beta-diol to androsterone. The substrate has been implicated in supporting gestation and modulating gamma-aminobutyric acid receptor activity. 17 beta HSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17 beta HSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5' untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17 beta HSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 or less adenosines (43% vs. 26%, P < 0.005). The C719T polymorphism is present in 15% of genomic DNA samples. Northern blot analysis showed high levels of 17 beta HSDXI expression in the pancreas, kidney, liver, lung, adrenal, ovary, and heart. Immunohistochemical staining for 17 beta HSDXI is strong in steroidogenic cells such as syncytiotrophoblasts, sebaceous gland, Leydig cells, and granulosa cells of the dominant follicle and corpus luteum. In the adrenal 17 beta HSDXI, staining colocalized with the distribution of 17 alpha-hydroxylase but was stronger in the mid to outer cortex. 17 beta HSDXI was also found in the fetus and increased after birth. Liver parenchymal cells and epithelium of the endometrium and small intestine also stained. Regulation studies in mouse Y1 cells showed that cAMP down-regulates 17 beta HSDXI enzymatic activity (40% vs. 32%, P < 0.05) and reduces gene expression to undetectable levels. All-trans-retinoic acid did not affect 17 beta HSDXI expression or activity, but addition of the retinoid together with cAMP significantly decreased activity over cAMP alone (32% vs. 23%, P < 0.05). Cloning and sequencing of the 17 beta HSDXI promoter identified the potential nuclear receptor steroidogenic factor-1 half-site TCCAAGGCCGG, and a cluster of three other potential steroidogenic factor-1 half-sites were found in the distal part of intron 1. Collectively, these results suggest a role for 17 beta HSDXI in androgen metabolism during steroidogenesis and a possible role in nonsteroidogenic tissues including paracrine modulation of 5 alpha-androstane-3 alpha, 17 beta-diol levels. 17 beta HSDXI could act by metabolizing compounds that stimulate steroid synthesis and/or by generating metabolites that inhibit it.

Diabetologia, 2010

Aims/hypothesis Excess accumulation of vascular extracellular matrix (ECM) is an important pathol... more Aims/hypothesis Excess accumulation of vascular extracellular matrix (ECM) is an important pathological process in cardiovascular diseases including diabetes-associated atherosclerosis. We explored how a recently identified molecule, cell division autoantigen 1 (CDA1), influences the profibrotic TGF-β pathway leading to vascular ECM accumulation. Methods Expression levels of genes encoding for CDA1, TGF-β and connective tissue growth factor (CTGF) were examined in aorta from Apoe −/− mice with or without diabetes. We used retroviral and adenoviral constructs to knockdown or overexpress Tspyl2, the gene encoding CDA1, in mouse vascular smooth muscle cells (VSMCs) with or without TGF-β treatment in order to demonstrate the role of CDA1 in TGF-β signalling. Results In vivo studies indicated that the mRNA levels of CDA1-encoding gene Tspyl2 and protein levels of CDA1 were elevated in the aorta of diabetic Apoe −/− mice, accompanied by increased levels of Tgf-β (also known as Tgfb1), Ctgf and ECM accumulation. In vitro studies in vascular cells showed that TGF-β treatment rapidly increased CDA1 protein levels, which then amplified TGF-β signalling leading to upregulation of ECM genes. Knockdown of CDA1-encoding gene Tspyl2 to reduce cellular CDA1 level markedly attenuated TGF-β-stimulated MAD homologue 3 (drosophila; SMAD3) phosphorylation and transcriptional activities. CDA1 overproduction increased and Tspyl2 knockdown decreased expression of TGF-β receptor type I, TβrI (also known as Tgfbr1), but not TGFβ receptor type II, TβrII (also known as Tgfbr2), providing a mechanism for CDA1's action in modulating TGF-β signalling. Knockdown of CDA1-encoding gene Tspyl2 also blocked the profibrotic effect of TGF-β in VSMCs. Conclusions/interpretation CDA1 plays an important role in vascular ECM accumulation by amplifying TGF-β signalling. This is critical for the profibrotic effect of TGF-β in the vasculature. CDA1 is therefore a potential target for attenuating vascular ECM accumulation caused by enhanced TGF-β action, as seen in diabetic atherosclerosis.

The American Journal of Cardiology, 2008

Kidney International, 2011

Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-b ... more Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-b (TGF-b) signaling in a number of cellular systems; here we found that its levels were elevated in the kidneys of two animal models of diabetic renal disease. The localization of CDA1 to tubular cells and podocytes in human kidney sections was similar to that seen in the rodent models. CDA1 small interfering RNA knockdown markedly attenuated, whereas its overexpression increased TGF-b signaling, modulating the expression of TGF-b, TGF-b receptors, connective tissue growth factor, collagen types I, III, IV, and fibronectin genes in HK-2 cells. CDA1 and TGF-b together were synergistic in stimulating TGF-b signaling and target gene expression. CDA1 knockdown effectively blocked TGF-b-stimulated expression of collagen genes. This was due to its ability to modulate the TGF-b type I, but not the type II, receptor, leading to increased phosphorylation of Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinase. Furthermore, the Smad3 inhibitor, SIS3, markedly attenuated the activities of CDA1 in stimulating TGF-b signaling as well as gene expression of collagens I, III, and IV. Thus, our in vitro and in vivo findings show that CDA1 has a critical role in TGF-b signaling in the kidney.