Barbara D. Smith

Investigative Urology: PGE sub 1 Suppresses the Induction of Collagen Synthesis by Transforming Growth Factor-beta sub 1 in Human Corpus Cavernosum Smooth Muscle

Collagen synthesis has been examined in primary cultures of human corpus cavernosum smooth muscle cells (HCCSMC), the major mesenchymal cell type of the corpus cavernosum. These cultures were grown from human surgical specimens and characterized by morphological and biochemical characteristics. These cells express mRNA for transforming growth factor-beta 1 (TGF-beta 1), a major regulator of extracellular matrix synthesis, as well as all three subtypes of TGF-beta receptors. Human corpus cavernosum smooth muscle cells primarily synthesize types I and III fibrillar collagen. Treatment of HCCSMC with exogenous TGF-beta 1 (80 pM.) induced a 2.5- to 4.5-fold increase in the synthesis of types I and III collagen and resulted in detectable levels of type V/XI collagen. Treatment of HCCSMC with the eicosanoid PGE1 in combination with TGF-beta 1 suppressed the induction of collagen synthesis by TGF-beta 1 in a dose-dependent manner with concomitant decreases in types I, III and V/XI collagen. The expression of TGF-beta 1 mRNA as well as types I and II TGF-beta receptors was induced by exogenous TGF-beta 1. Transforming growth factor-beta 1 mRNA induction was suppressed by PGE1. These data suggest that prostaglandins and TGF-beta 1 may play a key role in modulation of collagen synthesis in the corpus cavernosum, and in the regulation of fibrosis of the corpus cavernosum.

Nature of the collagen synthesized by a transplanted chondrosarcoma

Abstract A chondrosarcoma grown in rats was found to produce a single kind of collagen. This collagen was identified as a type II collagen, the cartilage specific protein.

Identification of collagen α1 (I) trimer and normal type I collagen in a polyoma virus-induced mouse tumor

A bone- and cartilage-forming mouse tumor, induced by transforming salivary epithelial cells with polyoma virus, contained large quantities of collagen. Two types of collagen molecules were isolated which had different solubilities in salt. One was type I collagen with a chain composition [al(I (~2 and the other was an unusual form of type I collagen with a chain composition [(xl(I)& This would appear to be the first in uiuo demonstration of (~1 type I trimer. A collagenous matrix is a ubiquitous constituent of solid tissues. Most cells (11,

Myocardin-related Transcription Factor-A Complexes Activate Type I Collagen Expression in Lung Fibroblasts

Background: Myofibroblasts are responsible for excessive collagen expression during scarring and fibrosis. Results: Myocardin-related transcription factor-A (MRTF-A) potentiates collagen overproduction through transcription complexes containing Sp1 and SRF on noncanonical DNA elements. Conclusion: MRTF-A promotes myofibroblast differentiation and collagen production through diverse molecular mechanisms. Significance: MRTF-A may be a molecular target in developing strategies to impede fibrosis. Pulmonary fibrosis is characterized by the excessive deposition of a collagen-rich extracellular matrix. The accumulation of collagen within the lung interstitium leads to impaired respiratory function. Furthermore, smooth muscle actin-positive myofibroblasts within the fibrotic lung contribute to disease progression. Because collagen and smooth muscle cell α-actin are coordinately expressed in the setting of fibrosis, the hypothesis was tested that specific transcriptional regulators of the myocardin family might also regulate collagen gene expression in myofibroblasts. Myocardin-related transcription factors (MRTFs), through their interaction with the serum-response factor (SRF) on CArG box regulatory elements (CC(A/T)6GG), are important regulators of myofibroblast differentiation. MRTF-A transactivated type I collagen gene reporters as much as 100-fold in lung myofibroblasts. Loss of functional MRTF-A using either a dominant negative MRTF-A isoform, shRNA targeting MRTF-A, or genetic deletion of MRTF-A in lung fibroblasts significantly disrupted type I collagen synthesis relative to controls. Analysis of the COL1A2 proximal promoter revealed a noncanonical CArG box (CCAAACTTGG), flanked by several Sp1 sites important for MRTF-A activation. Chromatin immunoprecipitation experiments confirmed the co-localization of MRTF-A, SRF, and Sp1 bound to the same region of the COL1A2 promoter. Mutagenesis of either the noncanonical CArG box or the Sp1 sites significantly disrupted MRTF-A activation of COL1A2. Together, our findings show that MRTF-A is an important regulator of collagen synthesis in lung fibroblasts and exhibits a dependence on both SRF and Sp1 function to enhance collagen expression.

Interferon-γ Induces Major Histocompatibility Class II Transactivator (CIITA), Which Mediates Collagen Repression and Major Histocompatibility Class II Activation by Human Aortic Smooth Muscle Cells

Chronic inflammation in atherosclerosis is responsible for plaque instability through alterations in extracellular matrix. Previously, we demonstrated that major histocompatibility class II (MHC II) transactivator (CIITA) in a complex with regulatory factor for X box 5 (RFX5) is a crucial protein mediating interferon (IFN)-γ–induced repression of collagen type I gene transcription in fibroblasts. This article demonstrates that, in smooth muscle cells (SMCs), IFN-γ dramatically increases the expression of CIITA isoforms III and IV, with no increase in expression of CIITA isoform I. Expression of CIITA III and IV correlates with decreased collagen type I and increased MHC II gene expression. Exogenous expression of CIITA I, III, and IV, in transiently transfected SMCs, represses collagen type I promoters (COL1A1 and COL1A2) and activates MHC II promoter. Levels of CIITA and RFX5 increase in the nucleus of cells treated with IFN-γ. Moreover, simvastatin lowers the IFN-γ–induced expression of RFX5 and MHC II in addition to repressing collagen expression. However, simvastatin does not block the IFN-γ–induced expression of CIITA III and IV, suggesting a CIITA-independent mechanism. This first demonstration that RFX5 and CIITA isoforms are expressed in SMCs after IFN-γ stimulation suggest that CIITA could be a key factor in plaque stability in atherosclerosis.

TNF-alpha upregulates the A2B adenosine receptor gene: the role of NAD(P)H oxidase 4

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-alpha) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A(2B) adenosine receptor (A(2B)AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-alpha. Here, we show a regulatory loop by which TNF-alpha upregulates the A(2B)AR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A(2B)AR, and Nox inhibitors dampen the effect of TNF-alpha. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A(2B)AR.

Angiotensinogen Genotype Affects Renal and Adrenal Responses to Angiotensin II in Essential Hypertension

Background—Renovascular and adrenal responses to infused angiotensin II (Ang II) are intermediate phenotypes that may indirectly reflect tissue renin-angiotensin system activity. We examine herein angiotensinogen (AGT) as a candidate gene to help elucidate potential mechanisms for previously reported AGT linkage and association studies. Methods and Results—Renal plasma flow and plasma aldosterone were measured before and after a 45-minute infusion of Ang II (3 ng · kg−1 · min−1) in 190 hypertensive patients who were on carefully controlled high- and low-salt diets. Reduced renal vascular (P =0.0002) and adrenal (P =0.002) responses to infused exogenous Ang II were associated with the AGT −6A allele. In multiple logistic regression, greater body mass index, lower basal renal plasma flow, and higher diastolic blood pressure together with AGT −6A genotype were associated with lower renal vascular response. In contrast, only male sex and AGT −6A genotype were associated with lower adrenal response. When both the renal and adrenal responses to Ang II were in the lowest tertile, the AGT −6AA genotype was present in 55.6%; in contrast, when both responses were in the upper 2 tertiles, the −6AA genotype was present in only 17.8% (P =0.001). Conclusions—A clear association between AGT genotype and response to infused Ang II was demonstrated for both the renal vasculature and the adrenal, consistent with the hypothesis that the AGT −6A genotype results in increased tissue expression of angiotensinogen and Ang II.

Collagen α1(I) Gene (COL1A1) Is Repressed by RFX Family

Collagen type I is composed of three polypeptide chains transcribed from two separate genes (COL1A1 and COL1A2) with different promoters requiring coordinate regulation. Our recent publications, centering on COL1A2 regulation, demonstrate that methylation in the first exon of COL1A2 at a regulatory factor for X box (RFX) site (at –1 to +20) occurs in human cancer cells and correlates with increased RFX1 binding and decreased collagen transcription (Sengupta, P. K., Erhlich, M., and Smith, B. D. (1999) J. Biol. Chem. 274, 36649–36655; Sengupta, S., Smith, E. M., Kim, K., Murnane, M. J., and Smith, B. D. (2003) Cancer Res. 63, 1789–1797). In normal cells, RFX5 complex along with major histocompatibility class II transactivator (CIITA) is induced by interferon-γ to occupy this site and repress collagen transcription (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J. Biol. Chem. 279, 41319–41332). In this paper, we demonstrate that COL1A1 has an RFX consensus binding site surrounding the transcription start site (–11 to +10) that contains three methylation sites rather than one in the COL1A2 gene RFX binding site. RFX1 interacts weakly with the unmethylated COL1A1 site, and binds with higher affinity to the methylated site. RFX1 represses the unmethylated COL1A1 less efficiently than COL1A2. COL1A1 promoter activity is sensitive to DNA methylation and the COL1A1 gene is methylated in human cancer cells with coordinately decreased collagen expression. The DNA methylation inhibitor, 5-aza-2′-deoxycytidine (aza-dC) increases collagen gene expression with time in human cancer cells. On the other hand, RFX5 interacts with both collagen type I genes with a similar binding affinity and represses both promoters equally in transient transfections. Two dominant negative forms of RFX5 activate both collagen genes coordinately. Finally, CIITA RNA interference experiments indicate that CIITA induction is required for interferon γ-mediated repression of both collagen type I genes.

A Methylation-responsive MDBP/RFX Site Is in the First Exon of the Collagen α2(I) Promoter

DNA methylation inhibits transcription driven by the collagen α2(I) promoter and the 5′ end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen α2(I) gene. Complex formation increased when the CpG site at +7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at +7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen α2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region.

Extracellular matrix synthesis in vascular disease： hypertension, and atherosclerosis

Extracellular matrix (ECM) within the vascular network provides both a structural and regulatory role. The ECM is a dynamic composite of multiple proteins that form structures connecting cells within the network. Blood vessels are distended by blood pressure and, therefore, require ECM components with elasticity yet with enough tensile strength to resist rupture. The ECM is involved in conducting mechanical signals to cells. Most importantly, ECM regulates cellular function through chemical signaling by controlling activation and bioavailability of the growth factors. Cells respond to ECM by remodeling their microenvironment which becomes dysregulated in vascular diseases such hypertension, restenosis and atherosclerosis. This review examines the cellular and ECM components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.