Myocardin-related transcription factor A regulates conversion of progenitors to beige adipocytes.

Adipose tissue is an essential regulator of metabolic homeostasis. In contrast with white adipose tissue, which stores excess energy in the form of triglycerides, brown adipose tissue is thermogenic, dissipating energy as heat via the unique expression of the mitochondrial uncoupling protein UCP1. A subset of UCP1+ adipocytes develops within white adipose tissue in response to physiological stimuli; however, the developmental origin of these "brite" or "beige" adipocytes is unclear. Here, we report the identification of a BMP7-ROCK signaling axis regulating beige adipocyte formation via control of the G-actin-regulated transcriptional coactivator myocardin-related transcription factor A, MRTFA. White adipose tissue from MRTFA(-/-) mice contains more multilocular adipocytes and expresses enhanced levels of brown-selective proteins, including UCP1. MRTFA(-/-) mice also show improved metabolic profiles and protection from diet-induced obesity and insulin resistance. Our study hence unravels a central pathway driving the development of physiologically functional beige adipocytes.

Penetrating colored pencil injury with Clostridium bifermentans pre-septal cellulitis: case report, literature review, and treatment algorithm.

The incidence of penetrating orbital injuries from writing instruments continues to rise in the pediatric population. Such injuries can cause significant visual morbidity and have a lifelong psychosocial impact. While the description of graphite pencil-related orbital trauma management is well demonstrated with over 40 reported cases, a lack of consistent management protocol for colored pencil-related injuries. Here, we report an inadvertent penetrating orbital colored pencil injury with progressive mechanical ptosis and pre-septal cellulitis necessitating urgent orbitotomy, debridement, and washout to reduce inflammatory and infectious burden. The wooden body serves as a nidus for polymicrobial infection, and the unique composition of colored pencil cores may lead to inflammatory processes that require vigilant multidisciplinary surgical and medical management reflected in our literature review.

Effect of Premature Tube Extrusion for Simple Congenital Nasolacrimal Duct Obstruction: A Comparison of Monocanalicular and Bicanalicular Intubation.

ABSTRACT: The objective of this article is to compare the incidence of premature dislocation of silicone tubes and the effect on treatment success between monocanalicular (MCI) and bicanalicular (BCI) intubation in pediatric patients with simple congenital nasolacrimal duct obstruction. Retrospective comparative case series of 108 eyes of 78 pediatric patients with simple congenital nasolacrimal duct obstruction who underwent probing with either BCI (n = 38 eyes) or MCI (n = 70 eyes) from 2017 to 2020. Premature tube extrusion was defined as any tube removed prior to the 3 month postoperative appointment. Success was defined as resolution of tearing 3 months post tube removal. Ages ranged from 10 months to 5.35 years (mean, 1.95 years; Standard deviation (SD), 0.91). Premature tube extrusion occurred in 15 eyes with BCI and 29 eyes with MCI. Success rates were not significantly different regardless of intubation type between the planned tube removal (90.6%) and the premature tube extrusion cohorts (84.1%), P = 0.89. There was no significant difference in treatment success between the planned tube removal (92.7% MCI, 87% BCI) and the premature tube extrusion cohorts (86.2% MCI, 80% BCI). Complications included 2 infections (1 MCI, 1 BCI) and 2 cases of tube related keratopathy (1 MCI, 1 BCI) that all resolved with tube removal. There were 2 BCI patients that presented to the emergency department for premature tube extrusion. Silicone intubation regardless of stent type is an effective treatment for simple congenital nasolacrimal duct obstruction. There was no significant difference in treatment success between tubes that extrude prematurely, and tubes removed at term based on type of intubation.

Aortic carboxypeptidase-like protein (ACLP) enhances lung myofibroblast differentiation through transforming growth factor ß receptor-dependent and -independent pathways.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFß, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFß signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFß receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFß receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFß signaling and promoting collagen expression through a TGFß receptor-independent pathway.

Myocardin-related transcription factor-A complexes activate type I collagen expression in lung fibroblasts.

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.

The effect of class II transactivator mutations on bleomycin-induced lung inflammation and fibrosis.

IFN-γ expression increases during the inflammatory response after bleomycin injury in mice. IFN-γ deficiency attenuates lung inflammation and fibrosis. Because IFN-γ stimulates class II transactivator (CIITA) expression, which activates major histocompatibility class (MHC) II and represses collagen expression, it was hypothesized that CIITA mediates IFN-γ action after bleomycin injury. To test this hypothesis, two CIITA mouse lines, one carrying a mutation of the leucine-rich region of CIITA (CIITA C-/-) and one with a deletion extending into the GTP-binding domain (CIITA G-/-), were used. IFN-γ treatment of lung cells isolated from both strains of mice induced mutant CIITA expression, which did not activate MHC II transcription. Collagen expression was similar in both mutant mouse strains and comparable to C57BL/6 (wild-type) mice. When mice were exposed to intratracheal bleomycin, both strains of CIITA mutant mice retained body weight and altered inflammation at 14 days after bleomycin injury compared with bleomycin-treated wild-type mice. However, there was no difference in fibrosis as judged by histology, mRNA, and protein expression of lungs. Bronchoalveolar lavage cells from CIITA C-/- and C57BL/6 lungs were examined at 3, 7, and 14 days after bleomycin injury. CD4 mRNA expression in bronchoalveolar lavage cells was down-regulated, whereas IL-4 and IL-10 expression was up-regulated, in CIITA C-/- mice, indicating a diminished, skewed Th2 response. The expression of IFN-γ was the same in all mice tested. Combined, our data suggest that CIITA mutations altered the immune response without affecting fibrosis.

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.

Interferon-gamma 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)-gamma-induced repression of collagen type I gene transcription in fibroblasts. This article demonstrates that, in smooth muscle cells (SMCs), IFN-gamma 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-gamma. Moreover, simvastatin lowers the IFN-gamma-induced expression of RFX5 and MHC II in addition to repressing collagen expression. However, simvastatin does not block the IFN-gamma-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-gamma stimulation suggest that CIITA could be a key factor in plaque stability in atherosclerosis.

Collagen and major histocompatibility class II expression in mesenchymal cells from CIITA hypomorphic mice.

Major histocompatibility class II (MHC II) transactivator (CIITA) is critical for interferon-gamma (IFN-gamma)-induced repression of collagen [Xu, Y., Wang, L., Buttice, G., Sengupta, P.K., Smith, B.D., 2004. Major histocompatibility class II transactivator (CIITA) mediates repression of collagen (COL1A2) transcription by interferon gamma (IFN-gamma). J. Biol. Chem. 279, 41319-41332] and activation of MHC II transcription. To better understand the role of CIITA and IFN-gamma induced repression of collagen, mesenchymal cells (lung fibroblasts, adventitial fibroblasts, and smooth muscle cells) were isolated from a CIITA deficient mouse (C2ta(tm1Ccum)). IFN-gamma induced MHC II expression and repressed collagen type I expression in all three cell types isolated from the wild type background. As expected, IFN-gamma treatment of cells isolated from CIITA deficient mice did not induce MHC II production or activate the MHC II promoter. Interestingly, collagen gene expression and promoter activity was similar to that of wild type. Moreover, IFN-gamma induced CIITA mRNA and a truncated form of CIITA protein in all cells isolated from CIITA deficient mice. Most importantly, truncated CIITA occupied the collagen alpha 2(I) gene (col1a2) transcription start site during IFN-gamma treatment, but it did not occupy the MHC II promoter as judged by chromatin immunoprecipitation assays. Exogenous expression of a similar truncated form of CIITA maintained its ability to repress col1a2 transcription, but lost its ability to activate MHC II gene transcription suggesting a role for the CIITA C-terminal domain in activation, but not repression. IFN-gamma induced primarily types I and IV CIITA isoforms in the mouse cells. All three isoforms of CIITA were capable of repressing col1a2 and activating MHC II gene transcription. These data suggest that the previously described CIITA knockout mouse carries a hypomorphic mutation, rather than a null mutation. The removal of the leucine rich region in CIITA blocks activation of MHC II without altering repression of collagen transcription.

DNA hypermethylation near the transcription start site of collagen alpha2(I) gene occurs in both cancer cell lines and primary colorectal cancers.

Collagen production plays a significant role in tumor development, especially in breast cancer, hepatocarcinomas, and colorectal carcinoma. However, collagen production is decreased during oncogenic transformation of cells in culture. This study demonstrates that methylation of the collagen alpha2(I) gene transcription start site occurs frequently in human cancer cell lines (9 of 10), including breast cancer cell lines (MCF-7 and Hs578T), hepatocellular carcinoma cell lines (SNU387, SNU449, SNU398, and PLC/PRF/5), a fibrosarcoma cell line (HT1080), and colorectal carcinoma cell lines (HCT116, SW480, and SW620). In addition, the collagen gene is more methylated in colorectal cancer tissues compared with normal mucosa. The increased DNA methylation of the collagen gene in cell lines is inversely correlated with collagen mRNA steady-state levels. Most importantly, treatment of fibrosarcoma or breast carcinoma cells with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, resulted in lower methylation and reactivation of the collagen gene in a dose-responsive manner. This is the first demonstration that the collagen alpha2(I) gene is methylated in multiple cancer cell lines correlating with loss of collagen expression and also methylated in primary cancer tissues. These data also suggest that methylation-induced repression of collagen transcription may be a frequent occurrence in cancer.

A Role of Myocardin Related Transcription Factor-A (MRTF-A) in Scleroderma Related Fibrosis.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.

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.

Regulating the activity of class II transactivator by posttranslational modifications: exploring the possibilities.

First identified as the master regulator of major histocompatibility complex II transcription, class II transactivator (CIITA) has since been implicated in a host of pathologies by modulating the transcription of multiple different genes. How CIITA caters to cell- and tissue-specific transcriptional needs is hotly debated and investigated. One of the possible mechanisms underlying spatiotemporal control of CIITA transcriptional activity is the posttranslational modification (PTM) machinery that refines certain amino acid residues of CIITA and hence alters its activity in response to specific cellular and environmental cues. This review discusses our current understanding of the PTM map of CIITA, how these modifications fine-tune its activity, and how the study of this area may lead to potential therapeutic strategies.

CIITA mediates interferon-gamma repression of collagen transcription through phosphorylation-dependent interactions with co-repressor molecules.

Previously, we have demonstrated that major histocompatibility class II trans-activator (CIITA) is crucial in mediating interferon-gamma (IFN-gamma)-induced repression of collagen type I gene transcription. Here we report that CIITA represses collagen transcription through a phosphorylation-dependent interaction between its proline/serine/threonine domain and co-repressor molecules such as histone deacetylase (HDAC2) and Sin3B. Mutation of a serine (S373A) in CIITA, within a glycogen synthase kinase 3 (GSK3) consensus site, decreases repression of collagen transcription by blocking interaction with Sin3B. In vitro phosphorylation of CIITA by GSK3 relies on a casein kinase I site three amino acids C-terminal to the GSK3 site in CIITA. Both GSK3 and casein kinase I inhibitors alleviate collagen repression and disrupt IFN-gamma-mediated recruitment of Sin3B and HDAC2 to the collagen start site. Therefore, we have identified the region within CIITA responsible for mediating IFN-gamma-induced inhibition of collagen synthesis.

Major histocompatibility class II transactivator expression in smooth muscle cells from A2b adenosine receptor knock-out mice: cross-talk between the adenosine and interferon-gamma signaling.

Atherosclerosis characterized by sustained inflammation and aberrant extracellular matrix alterations. Our previous investigation has defined major histocompatibility class II transactivator (CIITA) as a key factor in mediating these two processes in smooth muscle cells. Here, we demonstrate that CIITA and major histocompatibility class II expression are elevated in interferon-gamma (IFN-gamma)-treated smooth muscle cells from A2b adenosine receptor (A2bAR(-/-)) knock-out mice, as compared with wild type cells. An A2-type adenosine receptor agonist suppresses these effects of IFN-gamma in wild type cells, which can be blocked by an A2bAR-specific antagonist. We further identify that increased cellular cAMP levels are responsible for the down-regulation of CIITA expression and, hence, reduced IFN-gamma response as evidenced by the following data: 1) direct activation of adenylyl cyclase activity is both necessary and sufficient to suppress the IFN-gamma response; 2) inhibition of phosphodiesterase activity attenuates IFN-gamma induced transcription events; and 3) direct treatment with cAMP analog abrogates CIITA activation and IFN-gamma response. Therefore, our data establish possible cross-talk between the adenosine signaling through cAMP and IFN-gamma during regulation of CIITA expression.

Peroxisome proliferator-activated receptor gamma interacts with CIITA x RFX5 complex to repress type I collagen gene expression.

Recent reports demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, acts as a repressor of type I collagen synthesis. Our data demonstrate that exogenously expressed PPARgamma down-regulates collagen expression in a dose-responsive manner in human lung fibroblast cells. Silencing PPARgamma using lentiviruses expressing short hairpin RNAs partially reverses interferon-gamma (IFN-gamma)-induced repression and activates collagen mRNA levels. Previous studies indicate that IFN-gamma represses collagen gene expression and induces major histocompatibility complex II (MHC II) expression by activating the formation of a regulatory factor for X-box 5 (RFX5) complex with class II transactivator (CIITA). This report demonstrates that PPARgamma is within the RFX5.CIITA complex as judged by co-immunoprecipitation and DNA affinity precipitation studies. Most importantly, occupancy of PPARgamma on the collagen transcription start site and MHC II promoter increases with IFN-gamma treatment. The PPARgamma agonist, troglitazone, sensitizes the cells to IFN-gamma treatment by increasing recruitment of PPARgamma to collagen gene while repressing collagen expression, and these effects are blocked by the PPARgamma antagonist T0070907. PPARgamma may mediate IFN-gamma-stimulated collagen transcription down-regulation and MHC II up-regulation by interacting with CIITA as well as regulating CIITA expression. Therefore, PPARgamma is a critical target for investigations into therapeutics of diseases involving extracellular matrix remodeling and the immune response.

Regulatory factor for X-box family proteins differentially interact with histone deacetylases to repress collagen alpha2(I) gene (COL1A2) expression.

Our studies indicate that the regulatory factor for X-box (RFX) family proteins repress collagen alpha2(I) gene (COL1A2) expression (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2003) J. Biol. Chem. 278, 49134-49144; Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J. Biol. Chem. 279, 41319-41332). In this study, we examined the mechanism(s) underlying the repression of collagen gene by RFX proteins. Two members of the RFX family, RFX1 and RFX5, associate with distinct sets of co-repressors on the collagen transcription start site in vitro. RFX5 specifically interacts with histone deacetylase 2 (HDAC2) and the mammalian transcriptional repressor (mSin3B), whereas RFX1 preferably interacts with HDAC1 and mSin3A. HDAC2 cooperates with RFX5 to down-regulate collagen promoter activity, whereas HDAC1 enhances inhibition of collagen promoter activity by RFX1. Interferon-gamma promotes the recruitment of RFX5/HDAC2/mSin3B to the collagen transcription start site but decreases the occupancy by RFX1/mSin3A as manifested by chromatin immunoprecipitation assay. RFX1 binds to the methylated collagen sequence with much higher affinity than unmethylated sequence, recruiting more HDAC1 and mSin3A. The DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which inhibits DNA methylation, reduces RFX1/HDAC1 binding to the collagen transcription start site in chromatin immunoprecipitation assays. Finally, both RFX1 and RFX5 are acetylated in vivo. Trichostatin A stimulates the acetylation of RFX proteins and activates the collagen promoter activity. Collectively, our data strongly indicate two separate pathways for RFX proteins to repress collagen gene expression as follows: one for RFX5/HDAC2 in interferon-gamma-mediated repression, and the other for RFX1/HDAC1 in methylation-mediated collagen silencing.

Collagen alpha1(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-gamma 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 gamma-mediated repression of both collagen type I genes.

The RFX family interacts at the collagen (COL1A2) start site and represses transcription.

The transcription start site of the collagen alpha2(1) gene (COL1A2) has a sequence-specific binding site for a DNA methylation-responsive binding protein called regulatory factor for X-box 1 (RFX1) (Sengupta, P. K., Erhlich, M., and Smith, B. D. (1999) J. Biol. Chem. 274, 36649-36655). In this report, we demonstrate that RFX1 forms homodimers as well as heterodimers with RFX2 spanning the collagen transcription start site. Methylation at +7 on the coding strand increases RFX1 complex formation in gel shift assays. Methylation on the template strand, however, does not increase RFX1 complex formation. DNA from human fibroblasts contains minimal methylation on the coding strand (<4%) with variable methylation on the template strand. RFX1 acts as a repressor of collagen transcription as judged by in vitro transcription and co-transfection assays with an unmethylated collagen promoter-reporter construct. In addition, an RFX5 complex present in human fibroblasts interacts with the collagen RFX site, which is not sensitive to methylation. This is the first demonstration of RFX5 complex formation on a gene other than major histocompatibility complex (MHC) promoters. Also, RFX5 represses transcription of a collagen promoter-reporter construct in rat fibroblasts that have no detectable RFX5 complex formation or protein. RFX5 complex activates MHC II transcription by interacting with an interferon-gamma (IFN-gamma)-inducible protein, major histocompatibility class II trans-activator (CIITA). Collagen transcription is repressed by IFN-gamma in a dose-dependent manner in human but not in rat fibroblasts. IFN-gamma enhances RFX5 binding activity, and CIITA is present in the RFX5 complex of IFN-gamma-treated human fibroblasts. CIITA repressed collagen gene transcription more effectively in human fibroblasts than in rat fibroblasts, suggesting that the RFX5 complex may, in part, recruit CIITA protein to the collagen transcription start site. Thus the RFX family may be important repressors of collagen gene transcription through a RFX binding site spanning the transcription start site.

Interferon gamma repression of collagen (COL1A2) transcription is mediated by the RFX5 complex.

Interferon gamma (IFN-gamma) plays an important physiological role during inflammation by down-regulating collagen gene expression and activating major histocompatibility II (MHC-II) complex. The activation of MHC-II by IFN-gamma requires activation of a trimeric DNA binding transcriptional complex, RFX5 complex, containing RFXB (also called RFXANK or Tvl-1), RFXAP, as well as RFX5 protein. Previously, we demonstrated that RFX5 binds to the collagen transcription start site and represses collagen gene expression (Sengupta, P. K., Fargo, J., Smith, B. D. (2002) J. Biol. Chem. 277, 24926-24937). In this report, we have examined the role of RFXB and RFXAP proteins within the RFX5 complex to regulate collagen gene expression. The data show that all three RFX5 complex proteins are required for maximum repression. Expression of proteins with mutations known to be important for RFX5 complex formation does not repress collagen promoter activity. Two mutated forms of RFX5 act as dominant negative proteins activating collagen expression and reversing IFN-gamma down-regulation of collagen expression in human lung fibroblasts. IFN-gamma increases expression and nuclear translocation of RFX5. RFXB has a naturally occurring splice variant isoform (RFX SV). Interferon increases expression of the long form of RFXB and decreases expression of RFX SV with the same kinetics as collagen gene expression. Overexpression of the splice variant form reverses the IFN-gamma induced collagen repression in human lung fibroblasts. Finally, all three RFX5 complex proteins increase at the collagen transcription start site with IFN-gamma treatment using chromatin immunoprecipitation analysis. Thus, these studies suggest an important role for RFX5 complex in collagen repression.