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Biomedical subjects

M T Chin

Publications and source records attributed to M T Chin.

At least 19 recordsLinked to original sources
PubMed2001

Regulation of myogenic terminal differentiation by the hairy-related transcription factor CHF2.

We have recently cloned a novel basic helix-loop-helix factor, CHF2, that functions as a transcriptional repressor. To address its role in the regulation of myogenic terminal differentiation, we analyzed its expression pattern during C2C12 mouse myotube formation. In undifferentiated myoblasts, CHF2 is expressed at high levels. After induction of myotube formation in low serum, CHF2 expression is barely detectable at 3 days after induction. Myogenin expression, in contrast, peaks at 3 days. In transiently transfected 10T1/2 embryonic fibroblasts, CHF2 inhibited MyoD-dependent activation of the myogenin promoter in a dose-dependent fashion. Electrophoretic mobility shift analysis indicated that CHF2 inhibits the binding of the MyoD.E47 heterodimer to the E-box binding site. CHF2 also inhibited myogenic conversion of 10T1/2 cells by MyoD, as measured by skeletal myosin heavy chain protein expression. Coimmunoprecipitation analysis indicated that CHF2 forms a protein complex with MyoD. Mutational analysis of CHF2 indicated that the repression activity for both transcription and myogenic conversion mapped to a hydrophobic carboxyl-terminal region and did not require either the basic helix-loop-helix or YRPW motifs. Our data indicate that CHF2 functions as a transcriptional repressor of myogenesis by formation of an inactive heterodimeric complex with MyoD and likely plays an important role in muscle development.

Animals↗
PubMed2001

Down-regulation of high mobility group-I(Y) protein contributes to the inhibition of nitric-oxide synthase 2 by transforming growth factor-beta1.

The inducible isoform of nitric-oxide synthase (NOS2) catalyzes the production of nitric oxide (NO), which participates in the pathophysiology of systemic inflammatory diseases such as sepsis. NOS2 is transcriptionally up-regulated by endotoxin and inflammatory cytokines, and down-regulated by transforming growth factor (TGF)-beta1. Recently we have shown that high mobility group (HMG)-I(Y) protein, an architectural transcription factor, contributes to NOS2 gene transactivation by inflammatory mediators. The aim of the present study was to determine whether regulation of HMG-I(Y) by TGF-beta1 contributes to the TGF-beta1-mediated suppression of NOS2. By Northern blot analysis, we show that TGF-beta1 decreased cytokine-induced HMG-I(Y) mRNA levels in vascular smooth muscle cells and macrophages in vitro and in vivo. Western analysis confirmed the down-regulation of HMG-I(Y) protein by TGF-beta1. To determine whether the down-regulation of HMG-I(Y) contributed to a decrease in NOS2 gene transactivation by TGF-beta1, we performed cotransfection experiments. Overexpression of HMG-I(Y) was able to restore cytokine inducibility of the NOS2 promoter that was suppressed by TGF-beta1. The effect of TGF-beta1 on NOS2 gene transactivation was not related to a decrease in binding of HMG-I(Y) to the promoter of the NOS2 gene, but due to a decrease in endogenous HMG-I(Y) protein. These data provide the first evidence that cytokine-induced HMG-I(Y) can be down-regulated by TGF-beta1. This down-regulation of HMG-I(Y) contributes to the TGF-beta1-mediated decrease in NOS2 gene transactivation by proinflammatory stimuli.

Animals↗
PubMed2000

CLIF, a novel cycle-like factor, regulates the circadian oscillation of plasminogen activator inhibitor-1 gene expression.

The onset of myocardial infarction occurs frequently in the early morning, and it may partly result from circadian variation of fibrinolytic activity. Plasminogen activator inhibitor-1 activity shows a circadian oscillation and may account for the morning onset of myocardial infarction. However, the molecular mechanisms regulating this circadian oscillation remain unknown. Recent evidence indicates that basic helix-loop-helix (bHLH)/PAS domain transcription factors play a crucial role in controlling the biological clock that controls circadian rhythm. We isolated a novel bHLH/PAS protein, cycle-like factor (CLIF) from human umbilical vein endothelial cells. CLIF shares high homology with Drosophila CYCLE, one of the essential transcriptional regulators of circadian rhythm. CLIF is expressed in endothelial cells and neurons in the brain, including the suprachiasmatic nucleus, the center of the circadian clock. In endothelial cells, CLIF forms a heterodimer with CLOCK and up-regulates the PAI-1 gene through E-box sites. Furthermore, Period2 and Cryptochrome1, whose expression show a circadian oscillation in peripheral tissues, inhibit the PAI-1 promoter activation by the CLOCK:CLIF heterodimer. These results suggest that CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells. In addition, the results potentially provide a molecular basis for the morning onset of myocardial infarction.

ARNTL Transcription Factors↗
PubMed2000

Transforming growth factor-beta 1 inhibition of macrophage activation is mediated via Smad3.

Activated macrophages are critical cellular participants in inflammatory disease states. Transforming growth factor (TGF)-beta1 is a growth factor with pleiotropic effects including inhibition of immune cell activation. Although the pathway of gene activation by TGF-beta1 via Smad proteins has recently been elucidated, suppression of gene expression by TGF-beta1 remains poorly understood. We found that of Smad1-Smad7, Smad3 alone was able to inhibit expression of markers of macrophage activation (inducible nitric-oxide synthase and matrix metalloproteinase-12) following lipopolysaccharide treatment in gene reporter assays. Transient and constitutive overexpression of a dominant negative Smad3 opposed the inhibitory effect of TGF-beta1. Domain swapping experiments suggest that both the Smad MH-1 and MH-2 domains are required for inhibition. Mutation of a critical amino acid residue required for DNA binding in the MH-1 of Smad3 (R74A) resulted in the loss of inhibition. Transient overexpression of p300, an interactor of the Smad MH-2 domain, partially alleviated the inhibition by TGF-beta1/Smad3, suggesting that inhibition of gene expression may be due to increased competition for limiting amounts of this coactivator. Our results have implications for the understanding of gene suppression by TGF-beta1 and for the regulation of activated macrophages by TGF-beta1.

Animals↗
PubMed2000

Cardiovascular basic helix loop helix factor 1, a novel transcriptional repressor expressed preferentially in the developing and adult cardiovascular system.

We have cloned a cardiovascular-restricted basic helix-loop-helix factor that interacts with arylhydrocarbon receptor nuclear translocator (ARNT) in a yeast two-hybrid screen. Cardiovascular helix-loop-helix factor 1 (CHF1) is distantly related to the hairy family of transcriptional repressors. We analyzed its expression pattern during mouse embryo development. At day 8.5, the expression of CHF1 is first detected in the primitive ventricle of the primordial heart tube and persists throughout gestation. In rat hearts, this expression is down-regulated after birth, concurrent with terminal differentiation of cardiomyocytes. In the developing vasculature, CHF1 first appears in the dorsal aorta at day 9.0, which precedes the reported expression of smooth muscle cell markers, and persists into adulthood. In an in vitro system of smooth muscle cell differentiation, CHF1 mRNA was barely detectable in undifferentiated cells but was induced highly in differentiated smooth muscle cells. To determine whether CHF1 might affect the function of ARNT, we performed transfection studies. Co-transfection of CHF1 inhibited ARNT/EPAS1-dependent transcription by 85%, and this inhibition is dose-dependent. In electrophoretic mobility studies, CHF1 inhibited the binding of the ARNT/EPAS1 heterodimer to its target site. Our data suggest that CHF1 functions as a transcriptional repressor and may play an important role in cardiovascular development.

Animals↗
PubMed2000

Role of activating protein-1 and high mobility group-I(Y) protein in the induction of CD44 gene expression by interleukin-1beta in vascular smooth muscle cells.

CD44 is a multifunctional cell adhesion molecule that participates in pathological states such as inflammation and tumorigenesis. CD44 is induced on vascular smooth muscle cells after arterial wall injury and may mediate their proliferation and migration into the neointima during arteriosclerosis. We have demonstrated elsewhere that the proinflammatory cytokine interleukin (IL)-1beta up-regulates CD44 mRNA and protein expression in cultured rat aortic smooth muscle cells (RASMC) by increasing gene transcription. By transient transfection of 5'-deletion constructs into RASMC, we show in the present study that a conserved AP-1 site 110 base pairs from the transcription start site of the mouse CD44 promoter is important for basal activity. Mutation of the AP-1 site significantly reduced induction of promoter activity by IL-1beta, and electrophoretic mobility shift assays demonstrated that Fos and c-Jun were present in the CD44 AP-1 binding complex after IL-1beta stimulation. In addition, cotransfection of the architectural transcription factor high mobility group (HMG)-I(Y) protein with c-Fos and c-Jun markedly increased trans-activation of the CD44 promoter. Taken together, our studies demonstrate that AP-1 proteins are a central regulatory component used by IL-1beta to modulate expression of CD44 during an inflammatory response in vascular smooth muscle cells and that transcription of CD44 by AP-1 proteins is enhanced by HMG-I(Y). -Foster, L. C., Wiesel, P., Huggins, G. S, Pañares, R., Chin, M. T., Pellacani, A., Perrella, M. A. Role of activating protein-1 and high mobility group-I(Y) protein in the induction of CD44 gene expression by interleukin-1beta in vascular smooth muscle cells.

Animals↗
PubMed1999

Characterization of the mUBC9-binding sites required for E2A protein degradation.

Mammalian Ubc9 (mUbc9) is required for rapid degradation of the E2A proteins E12 and E47 by the ubiquitin-proteasome system. We have shown elsewhere that mUbc9 interacts with amino acids 477-530 of E12/E47. Here we test the hypothesis that this region, rich in proline, glutamic acid, serine, and threonine (PEST) residues, serves as the E2A protein degradation domain (DD). An E2A protein lacking this region, E47Delta(478-531), was significantly more stable than wild-type E47(half-life of more than 6 h versus 55 min). Deletion of the E2A DD had no effect on the E-box-binding and transcriptional activity of E47. We mapped two discreet mUbc9-interacting regions within the E2A DD: amino acids 476-494 and 505-513. E2A(505-513) interacted with mUbc9 but not with human Ubc5, MyoD, Id3, or the polymyositis-scleroderma autoantigen. Substitution of the E2A(505-513) central hydrophobic residues with basic residues abolished interaction with mUbc9. Also, full-length E47 lacking the second mUbc9-interacting region was significantly more stable than wild-type E47. Reintroduction of the E2A DD into the long-lived, naturally occurring chimeric oncoprotein E2A-HLF (hepatic leukemic factor) destabilized it, suggesting that this domain can transfer a degradation signal to a heterologous protein. E2A-HLF-DD chimeric protein was stabilized by the proteasome inhibitor LLNL, indicating the role of the ubiquitin-proteasome system mediating degradation through the E2A degradation domain. Our experiments indicate that the E2A DD mediates E2A protein interactions with the ubiquitin-proteasome system and that the E2A DD is required for metabolism of these widely expressed proteins.

3T3 Cells↗
PubMed1999

High mobility group-I(Y) protein facilitates nuclear factor-kappaB binding and transactivation of the inducible nitric-oxide synthase promoter/enhancer.

Nitric oxide (NO), a free radical gas whose production is catalyzed by the enzyme NO synthase, participates in the regulation of multiple organ systems. The inducible isoform of NO synthase (iNOS) is transcriptionally up-regulated by inflammatory stimuli; a critical mediator of this process is nuclear factor (NF)-kappaB. Our objective was to determine which regulatory elements other than NF-kappaB binding sites are important for activation of the iNOS promoter/enhancer. We also wanted to identify transcription factors that may be functioning in conjunction with NF-kappaB (subunits p50 and p65) to drive iNOS transcription. Deletion analysis of the iNOS promoter/enhancer revealed that an AT-rich sequence (-61 to -54) downstream of the NF-kappaB site (-85 to -76) in the 5'-flanking sequence was important for iNOS induction by interleukin-1beta and endotoxin in vascular smooth muscle cells. This AT-rich sequence, corresponding to an octamer (Oct) binding site, bound the architectural transcription factor high mobility group (HMG)-I(Y) protein. Electrophoretic mobility shift assays showed that HMG-I(Y) and NF-kappaB subunit p50 bound to the iNOS promoter/enhancer to form a ternary complex. The formation of this complex required HMG-I(Y) binding at the Oct site. The location of an HMG-I(Y) binding site typically overlaps that of a recruited transcription factor. In the iNOS promoter/enhancer, however, HMG-I(Y) formed a complex with p50 while binding downstream of the NF-kappaB site. Furthermore, overexpression of HMG-I(Y) potentiated iNOS promoter/enhancer activity by p50 and p65 in transfection experiments, suggesting that HMG-I(Y) contributes to the transactivation of iNOS by NF-kappaB.

Animals↗
PubMed1999

Induction of high mobility group-I(Y) protein by endotoxin and interleukin-1beta in vascular smooth muscle cells. Role in activation of inducible nitric oxide synthase.

Nonhistone chromosomal proteins of the high mobility group (HMG) affect the transcriptional regulation of certain mammalian genes. For example, HMG-I(Y) controls cytokine-mediated promoters that require transcription factors, such as nuclear factor-kappaB, for maximal expression. Even though a great deal is known about how HMG-I(Y) facilitates expression of other genes, less is known about the regulation of HMG-I(Y) itself, especially in cells in primary culture. Therefore we investigated the effect of endotoxin and the cytokine interleukin-1beta on HMG-I(Y) expression in vascular smooth muscle cells. Induction of HMG-I(Y) peaked after 48 h of interleukin-1beta stimulation (6.2-fold) in cells in primary culture, and this increase in mRNA corresponded to an increase in HMG-I(Y) protein. Moreover, immunohistochemical staining revealed a dramatic increase in HMG-I(Y) protein expression in vascular smooth muscle cells after endotoxin stimulation in vivo. This increase in HMG-I(Y) expression (both in vitro and in vivo) mirrored an up-regulation of inducible nitric oxide synthase, a cytokine-responsive gene. The functional significance of this coinduction is underscored by our finding that HMG-I(Y) potentiated the response of inducible nitric oxide synthase to nuclear factor-kappaB transactivation. Taken together, these studies suggest that induction of HMG-I(Y), and subsequent transactivation of iNOS, may contribute to a reduction in vascular tone during endotoxemia and other systemic inflammatory processes.

Animals↗
PubMed1999

Induction of high mobility group I architectural transcription factors in proliferating vascular smooth muscle in vivo and in vitro.

Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arteriosclerosis. Architectural transcription factors of the high mobility group (HMG)-I family have been implicated in the control of cell proliferation and gene expression. We studied the pattern of HMG-I mRNA and protein expression in proliferating VSMCs. HMG-I(Y) and HMGI-C mRNAs were barely detectable by Northern analysis in samples prepared from uninjured rat carotid arteries. In contrast, these mRNAs were induced dramatically in carotid arteries 2 and 5-6 days after balloon injury. By in situ hybridization at 6 days after injury, the induced mRNAs localized to smooth muscle cells of the developing neointima, and immunocytochemical analysis showed that HMG-I(Y) protein was expressed in the nuclei of these cells. To confirm this association between HMG-I protein induction and cell growth, we assessed HMG-I(Y) and HMGI-C mRNA expression in rat aortic smooth muscle cells (RASMCs) in primary culture. The HMG-I mRNAs were barely detectable in quiescent RASMCs but were induced markedly by serum stimulation. This induction of mRNA by serum was time dependent and peaked at 9 h. Western blot analysis confirmed that HMG-I(Y) protein induction also occurred in vitro. To our knowledge, this is the first demonstration of induction of HMG-I protein expression in proliferating RASMCs in vivo and in vitro. This demonstration suggests that the HMG-I proteins may play an important role in smooth muscle cell proliferation.

Animals↗
PubMed1998

Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system.

Proteins of the LIM family are critical regulators of development and differentiation in various cell types. We have described the cloning of cysteine-rich protein 2/smooth muscle LIM protein (CRP2/SmLIM), a LIM-only protein expressed in differentiated vascular smooth muscle cells. As a first step toward understanding the potential functions of CRP2/SmLIM, we analyzed its expression after gastrulation in developing mice and compared the expression of CRP2/SmLIM with that of the other 2 members of the CRP subclass, CRP1 and CRP3/MLP. In situ hybridization in whole-mount and sectioned embryos showed that CRP2/SmLIM was expressed in the sinus venosus and the 2 cardiac chambers at embryonic day 9. Vascular expression of CRP2/SmLIM was first seen at embryonic day 10. At subsequent time points, CRP2/SmLIM expression decreased in the heart but remained high in the vasculature. CRP1 was expressed both in vascular and nonvascular tissues containing smooth muscle cells, whereas CRP3/MLP was expressed only in tissues containing striated muscle. These patterns of expression were maintained in the adult animal and suggest an important role for this gene family in the development of smooth and striated muscle.

Age Factors↗
PubMed1998

Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation.

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.

Adult↗
PubMed1998

Molecular cloning, characterization, and promoter analysis of the mouse Crp2/SmLim gene. Preferential expression of its promoter in the vascular smooth muscle cells of transgenic mice.

Several members of the LIM protein family have important roles in development and differentiation. We recently isolated a rat cDNA encoding a new member of this family, CRP2/SmLIM, that contains two LIM domains and is expressed preferentially in vascular smooth muscle cells (VSMC). To study the molecular mechanisms that regulate VSMC-specific transcription of the Crp2/SmLim gene, we cloned the cDNA and gene of mouse Crp2/SmLim. Mouse Crp2/SmLim is a single copy gene of six exons and five introns spanning approximately 20 kilobases of genomic DNA. By 5'-rapid amplification of cDNA ends and S1 nuclease protection assay, we determined that the transcription start site is an A residue 80 base pairs 5' of the translation initiation codon. A TATA-like sequence is located 27 base pairs 5' of the transcription start site, and there are potential cis-acting elements (GATA, Sp1, AP-2, E box, CCAC box, and GArC motif) in the 5'-flanking sequence. In transient transfection assays in rat aortic smooth muscle cells in primary culture, 5 kilobases of the Crp2/SmLim 5'-flanking sequence generated a high level of luciferase reporter gene activity. By deletion analysis and gel mobility shift assay, we found that the region between bases -74 and -39 of this 5 kilobase DNA fragment binds Sp1 and confers basal promoter activity in the Crp2/SmLim gene. In vitro, the 5-kilobase fragment was active in multiple cell types. In vivo, however, the 5-kilobase fragment directed high level expression of the lacZ reporter gene preferentially in the VSMC of transgenic mice, indicating the presence of VSMC-specific element(s) in this fragment.

Amino Acid Sequence↗
PubMed1998

Enhancement of serum-response factor-dependent transcription and DNA binding by the architectural transcription factor HMG-I(Y).

The mechanisms by which HMG-I proteins regulate cell growth are unknown, and their effects on gene expression have only been partially elucidated. We explored the potential interaction between HMG-I proteins and serum-response factor (SRF), a member of the MADS-box family of transcription factors. In cotransfection experiments, HMG-I(Y) potentiated SRF-dependent activation (by more than 5-fold) of two distinct SRF-responsive promoters, c-fos and the smooth muscle-specific gene SM22alpha. This effect was also observed with a heterologous promoter containing multiple copies of the CC(A/T)6GG (CArG) box. HMG-I proteins bound specifically to the CArG boxes of c-fos and SM22alpha in gel mobility shift analysis and enhanced binding of SRF to these CArG boxes. By chelating peptide-immobilized metal affinity chromatography, we mapped the domain of HMG-I(Y) that interacts with SRF to amino acids 50-81, a region that does not bind specifically to DNA in electrophoretic mobility shift assays even though it includes the third AT-hook DNA-binding domain. Surprisingly, HMG-I(Y) mutants that failed to bind DNA still enhanced SRF binding to DNA and SRF-dependent transcription. In contrast, deletion of the HMG-I(Y) 50-81 domain that bound SRF prevented enhancement of transcription. To our knowledge, this is the first report of an HMG-I protein interacting with a MADS-box transcription factor. Our observations suggest that members of the HMG-I family play an important role in SRF-dependent transcription and that their effect is mediated primarily by a protein-protein interaction.

Animals↗
PubMed1998

In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells.

The change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent induction of the SMC markers smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM22alpha, and APEG-1. The activity of the SM22alpha promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the wild-type SM22alpha promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we identified new DNA-protein complexes in nuclear extracts prepared from differentiated Monc-1 cells. One of the new complexes contained serum response factor. This Monc-1 to SMC model should facilitate the identification of nodal regulators of smooth muscle development and differentiation.

Animals↗
PubMed1998

Human EZF, a Krüppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains.

Members of the erythroid Krüppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the beta-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the beta-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.

Amino Acid Sequence↗
PubMed1998

Pregnancy and delivery in a patient with recent peripartum cardiomyopathy.

We present a case of successful pregnancy and delivery in a patient with peripartum cardiomyopathy who conceived only 3 months after a vaginal delivery complicated by peripartum cardiomyopathy. Following the onset of labor, an arterial pressure catheter and pulmonary artery catheter were placed. A combined epidural spinal technique was performed without difficulty for labor analgesia. After a few hours of labor, the fetal heart rate tracing revealed repetitive variable decelerations, and a decision was made to proceed with operative delivery. The mother's hemodynamic changes are presented, and the specifics of the anesthetic care are outlined.

Journal Article↗