Changes in nascent chromatin structure regulate activation of the pro-fibrotic transcriptome and myofibroblast emergence in organ fibrosis.

Cell reprogramming to a myofibroblast responsible for the pathological accumulation of extracellular matrix is fundamental to the onset of fibrosis. Here, we explored how condensed chromatin structure marked by H3K72me3 becomes modified to allow for activation of repressed genes to drive emergence of myofibroblasts. In the early stages of myofibroblast precursor cell differentiation, we discovered that H3K27me3 demethylase enzymes UTX/KDM6B creates a delay in the accumulation of H3K27me3 on nascent DNA revealing a period of decondensed chromatin structure. This period of decondensed nascent chromatin structure allows for binding of pro-fibrotic transcription factor, Myocardin-related transcription factor A (MRTF-A) to nascent DNA. Inhibition of UTX/KDM6B enzymatic activity condenses chromatin structure, prevents MRTF-A binding, blocks activation of the pro-fibrotic transcriptome, and results in an inhibition of fibrosis in lens and lung fibrosis models. Our work reveals UTX/KDM6B as central coordinators of fibrosis, highlighting the potential to target its demethylase activity to prevent organ fibrosis.

Keloid disorder: Fibroblast differentiation and gene expression profile in fibrotic skin diseases.

Keloid disorder, a group of fibroproliferative skin diseases, is characterized by unremitting accumulation of the extracellular matrix (ECM) of connective tissue, primarily collagen, to develop cutaneous tumors on the predilection sites of skin. There is a strong genetic predisposition for keloid formation, and individuals of African and Asian ancestry are particularly prone. The principal cell type responsible for ECM accumulation is the myofibroblast derived from quiescent resident skin fibroblasts either through trans-differentiation or from keloid progenitor stem cells with capacity for multi-lineage differentiation and self-renewal. The biosynthetic pathways leading to ECM accumulation are activated by several cytokines, but particularly by TGF-ß signalling. The mechanical properties of the cellular microenvironment also play a critical role in the cell's response to TGF-ß, as demonstrated by culturing of fibroblasts derived from keloids and control skin on substrata with different degrees of stiffness. These studies also demonstrated that culturing of fibroblasts on tissue culture plastic in vitro does not reflect their biosynthetic capacity in vivo. Collectively, our current understanding of the pathogenesis of keloids suggests a complex network of interacting cellular, molecular and mechanical factors, with distinct pathways leading to myofibroblast differentiation and activation. Keloids can serve as a model system of fibrotic diseases, a group of currently intractable disorders, and deciphering of the critical pathogenetic steps leading to ECM accumulation is expected to identify targets for pharmacologic intervention, not only for keloids but also for a number of other, both genetic and acquired, fibrotic diseases.

Trametinib prevents mesothelial-mesenchymal transition and ameliorates abdominal adhesion formation.

BACKGROUND: Intra-abdominal adhesions are a major cause of morbidity after abdominal or gynecologic surgery. However, knowledge about the pathogenic mechanism(s) is limited, and there are no effective treatments. Here, we investigated a mouse model of bowel adhesion formation and the effect(s) of an Federal Drug Administration-approved drug (trametinib) in preventing adhesion formation. MATERIALS AND METHODS: C57BL/6 mice were used to develop a consistent model of intra-abdominal adhesion formation by gentle cecal abrasion with mortality rates of <10%. Adhesion formation was analyzed histologically and immunochemically to characterize the expression of pro-fibrotic marker proteins seen in pathologic scaring and included alpha smooth muscle actin (αSMA) and fibronectin EDA (FNEDA) which arises from alternative splicing of the fibronectin messenger RNA resulting in different protein isoforms. Trichrome staining assessed collagen deposition. Quantitative polymerase chain reaction analysis of RNA isolated from adhesions by laser capture microscopy was carried out to assess pro-fibrotic gene expression. To block adhesion formation, trametinib was administered via a subcutaneous osmotic pump. RESULTS: Adhesions were seen as early as post-operative day 1 with extensive adhesions being formed and vascularized by day 5. The expression of the FNEDA isoform occurred first with subsequent expression of αSMA and collagen. The drug trametinib was chosen for in vivo studies because it effectively blocked the mesothelial to mesenchymal transition of rat mesothelium. Trametinib, at the highest dose used (3 mg/kg/d), prevented adhesion formation while at lower doses, adhesions were usually limited, as evidenced by the presence of FNEDA isoform but not αSMA. CONCLUSIONS: Cecal abrasion in mice is a reliable model to study abdominal adhesions, which can be ameliorated using the MEK1/2 inhibitor trametinib. While blocking adhesion formation at the cell and molecular levels, trametinib, at the therapeutic doses utilized, did not impair the wound healing at the laparotomy site.

Therapeutic Approaches to Radiation-Induced Fibrosis.

Radiation induced fibrosis (RIF) is a common morbidity in patients being treated for cancer with radiation. Off-target effects result in intense inflammatory responses which ultimately results in the generation of extracellular matrix (ECM) producing myofibroblasts which mediate a progressive fibrosis resulting in scarring and organ and tissue dysfunction. Unfortunately, currently, there are no effective therapies to block the excess accumulation of ECM. We have previously reported on the use of trametinib, a MEK inhibitor, to essentially block the formation of abdominal adhesions in a mouse model of cecal abrasion. Using this drug in the mouse model, the complete trans-differentiation of precursor cells into ECM-producing myofibroblasts was blocked. Trametinib is a potentially powerful drug to thwart organ and tissue fibrosis in RIF because it has a potential dual function in that it may block RIF as well as prevent radiation-resistance. Given the intractability of RIF, trametinib should be considered for more extensive testing.

Human Fibrotic Diseases: Current Challenges in Fibrosis Research.

Human fibrotic diseases constitute a major health problem worldwide owing to the large number of affected individuals, the incomplete knowledge of the fibrotic process pathogenesis, the marked heterogeneity in their etiology and clinical manifestations, the absence of appropriate and fully validated biomarkers, and, most importantly, the current void of effective disease-modifying therapeutic agents. The fibrotic disorders encompass a wide spectrum of clinical entities including systemic fibrotic diseases such as systemic sclerosis (SSc), sclerodermatous graft vs. host disease, and nephrogenic systemic fibrosis, as well as numerous organ-specific disorders including radiation-induced fibrosis and cardiac, pulmonary, liver, and kidney fibrosis. Although their causative mechanisms are quite diverse and in several instances have remained elusive, these diseases share the common feature of an uncontrolled and progressive accumulation of fibrotic tissue in affected organs causing their dysfunction and ultimate failure. Despite the remarkable heterogeneity in the etiologic mechanisms responsible for the development of fibrotic diseases and in their clinical manifestations, numerous studies have identified activated myofibroblasts as the common cellular element ultimately responsible for the replacement of normal tissues with nonfunctional fibrotic tissue. Critical signaling cascades, initiated primarily by transforming growth factor-ß (TGF-ß), but also involving numerous cytokines and signaling molecules which stimulate profibrotic reactions in myofibroblasts, offer potential therapeutic targets. Here, we briefly review the current knowledge of the molecular mechanisms involved in the development of tissue fibrosis and point out some of the most important challenges to research in the fibrotic diseases and to the development of effective therapeutic approaches for this often fatal group of disorders. Efforts to further clarify the complex pathogenetic mechanisms of the fibrotic process should be encouraged to attain the elusive goal of developing effective therapies for these serious, untreatable, and often fatal disorders.

Keloids: The paradigm of skin fibrosis - Pathomechanisms and treatment.

Keloids, fibroproliferative dermal tumors with effusive accumulation of extracellular matrix (ECM) components, particularly collagen, result from excessive expression of growth factors and cytokines. The etiology of keloids is unknown but they occur after dermal injury in genetically susceptible individuals, and they cause both physical and psychological distress for the affected individuals. Several treatment methods for keloids exist, including the combination therapy of surgical excision followed by intralesional steroid therapy, however, they have high recurrence rate regardless of the current treatment method. Improved understanding of the pathomechanisms leading to keloid formation will hopefully identify pathways that serve as specific targets to improve therapy for this devastating, currently intractable, disorder.

Pathobiological mechanisms of peritoneal adhesions: The mesenchymal transition of rat peritoneal mesothelial cells induced by TGF-ß1 and IL-6 requires activation of Erk1/2 and Smad2 linker region phosphorylation.

Peritoneal adhesions, primarily caused by surgical procedures, are the leading cause of pelvic pain, bowel obstruction, and infertility. TGF-ß1 and IL-6 have been found to be elevated in the peritoneal fluid of patients during/after abdominal surgery. However, it remains to be determined whether these cytokines interact and facilitate adhesion formation by promoting mesothelial to mesenchymal transition (MMT). In the present study, isolated rat peritoneal mesothelial cells were treated with TGF-ß1 and/or IL-6 which elicited MMT as determined by morphologic and biochemical techniques. During this transition, cellular morphology changed from that of cobblestone polygonal cells to elongated/spindle-shaped fibroblast-like cells. There was decreased expression of genes characteristic of mesothelial cells, such as E-cadherin, and increased expression of genes characteristic of the myofibroblast phenotype, including α-smooth muscle actin and the EDA form of fibronectin, both of which appear to mediate the transfer of force to the extracellular matrix. Partial characterization of relevant signaling pathways identified Erk1/2 activation, which was enhanced by combined TGF-ß1/IL-6 administration, as a crucial necessary factor in the transition. Erk1/2 activation as well as the phosphorylation of the linker region of Smad2 and MMT could be blocked by the MEK inhibitor, U0126, suggesting that such activation may be a potential pharmaceutical target to prevent MMT. In addition, the phenotypic transition could be prevented by hydrocortisone.

New frontiers in fibrotic disease therapies: The focus of the Joan and Joel Rosenbloom Center for Fibrotic Diseases at Thomas Jefferson University.

Fibrotic diseases constitute a world-wide major health problem, but research support remains inadequate in comparison to the need. Although considerable understanding of the pathogenesis of fibrotic reactions has been attained, no completely effective therapies exist. Although fibrotic disorders are diverse, it is universally appreciated that a particular cell type with unique characteristics, the myofibroblast, is responsible for replacement of functioning tissue with non-functional scar tissue. Understanding the cellular and molecular mechanisms responsible for the creation of myofibroblasts and their activities is central to the development of therapies. Critical signaling cascades, initiated primarily by TGF-ß, but also involving other cytokines which stimulate pro-fibrotic reactions in the myofibroblast, offer potential therapeutic targets. However, because of the multiplicity and complex interactions of these signaling pathways, it is very unlikely that any single drug will be successful in modifying a major fibrotic disease. Therefore, we have chosen to examine the effectiveness of administration of several drug combinations in a mouse pneumoconiosis model. Such treatment proved to be effective. Because fibrotic diseases that tend to be chronic, are difficult to monitor, and are patient variable, implementation of clinical trials is difficult and expensive. Therefore, we have made efforts to identify and validate non-invasive biomarkers found in urine and blood. We describe the potential utility of five such markers: (i) the EDA form of fibronectin (Fn(EDA)), (ii) lysyl oxidase (LOX), (iii) lysyl oxidase-like protein 2 (LoxL2), (iv) connective tissue growth factor (CTGF, CCNII), and (v) the N-terminal propeptide of type III procollagen (PIIINP).

Absence of γ-sarcoglycan alters the response of p70S6 kinase to mechanical perturbation in murine skeletal muscle.

BACKGROUND: The dystrophin glycoprotein complex (DGC) is located at the sarcolemma of muscle fibers, providing structural integrity. Mutations in and loss of DGC proteins cause a spectrum of muscular dystrophies. When only the sarcoglycan subcomplex is absent, muscles display severe myofiber degeneration, but little susceptibility to contractile damage, suggesting that disease occurs not by structural deficits but through aberrant signaling, namely, loss of normal mechanotransduction signaling through the sarcoglycan complex. We extended our previous studies on mechanosensitive, γ-sarcoglycan-dependent ERK1/2 phosphorylation, to determine whether additional pathways are altered with the loss of γ-sarcoglycan. METHODS: We examined mechanotransduction in the presence and absence of γ-sarcoglycan, using C2C12 myotubes, and primary cultures and isolated muscles from C57Bl/6 (C57) and γ-sarcoglycan-null (γ-SG(-/-)) mice. All were subjected to cyclic passive stretch. Signaling protein phosphorylation was determined by immunoblotting of lysates from stretched and non-stretched samples. Calcium dependence was assessed by maintaining muscles in calcium-free or tetracaine-supplemented Ringer's solution. Dependence on mTOR was determined by stretching isolated muscles in the presence or absence of rapamycin. RESULTS: C2C12 myotube stretch caused a robust increase in P-p70S6K, but decreased P-FAK and P-ERK2. Neither Akt nor ERK1 were responsive to passive stretch. Similar but non-significant trends were observed in C57 primary cultures in response to stretch, and γ-SG(-/-) cultures displayed no p70S6K response. In contrast, in isolated muscles, p70S6K was mechanically responsive. Basal p70S6K activation was elevated in muscles of γ-SG(-/-) mice, in a calcium-independent manner. p70S6K activation increased with stretch in both C57 and γ-SG(-/-) isolated muscles, and was sustained in γ-SG(-/-) muscles, unlike the transient response in C57 muscles. Rapamycin treatment blocked all of p70S6K activation in stretched C57 muscles, and reduced downstream S6RP phosphorylation. However, even though rapamycin treatment decreased p70S6K activation in stretched γ-SG(-/-) muscles, S6RP phosphorylation remained elevated. CONCLUSIONS: p70S6K is an important component of γ-sarcoglycan-dependent mechanotransduction in skeletal muscle. Our results suggest that loss of γ-sarcoglycan uncouples the response of p70S6K to stretch and implies that γ-sarcoglycan is important for inactivation of this pathway. Overall, we assert that altered load-sensing mechanisms exist in muscular dystrophies where the sarcoglycans are absent.

Mechanosensitive release of adenosine 5'-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain.

As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.

Recombinant insulin-like growth factor-1 activates satellite cells in the mouse urethral rhabdosphincter.

BACKGROUND: The goal of this study is to demonstrate the efficacy of a new method for the treatment of urinary incontinence by stimulation of urethral rhabdosphincter satellite cells. We show that satellite cells do exist in the sphincter muscle of retired male mice breeders by staining for c-Met, a satellite cell specific protein. Once activated by recombinant mouse Insulin-like Growth Factor-1(rIgf-1), the satellite cells develop into muscle cells within the rhabdosphincter thereby potentially strengthening it. METHODS: 20 µl (1 µg/µl) of rIgf-1 was surgically injected directly into the urethral wall of retired male mouse breeders. Mice injected with phosphate buffered saline (PBS) were used as controls. 4 weeks later, urethras were harvested and serially-sectioned through the sphincter for routine hematoxylin-eosin staining as well as immunohistochemical staining with satellite cell specific anti-c-Met antibody and proliferation specific anti-Ki-67 antibody. RESULTS: Anti-c-Met antibody positive cells (c-Met+) were identified in the rhabdosphincter. c-Met+ cells increased by 161.8% relative to controls four weeks after rIGF-1 injection. Anti- Ki-67 antibody positive cells were identified and characterized as cells with centrally located nuclei in striated muscle bundles of rIGF-1 treated animals. CONCLUSIONS: Satellite cells in the mouse rhabdosphincter can be activated by rIGF-1 treatment, which subsequently are incorporated into existing skeletal muscle bundles. Using this approach, the rhabdosphincter can be induced to regenerate and potentially strengthen via satellite cell activation and likely improve urinary continence.

Urinary diversion results in marked decreases in proliferation and apoptosis in fetal bladder.

PURPOSE: We sought to determine if bladder cycling is required for remodeling during fetal development. MATERIALS AND METHODS: For this study 5 fetal sheep bladders were harvested after 2 weeks of urinary diversion, initiated at approximately 90 days of gestation. Six unoperated sheep bladders of approximately 105 days of gestational age were used as controls. Dividing cells and cells undergoing apoptosis were quantified by using Ki-67 antibody and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) assay, respectively. In addition, expression of the antiapoptosis gene, Bcl2, and cell division control protein 42 were quantified by real-time polymerase chain reaction. RESULTS: The thickness of bladder tissue layers is dramatically altered as a consequence of urinary diversion (defunctionalization). The percentage of Ki-67 and TUNEL positive cells in control bladders was 5.8% and 47.1%, respectively. However, in diverted bladders apoptosis and cell mitosis were significantly decreased with essentially 0% of Ki-67 and TUNEL positive cells per microscope field in the mucosa and detrusor muscle layers. In addition, relative mRNA expression of antiapoptotic gene Bcl2 was significantly lower in control than in diverted bladder tissue, while Cdc42 was significantly higher in the detrusor but not in the lamina propria. CONCLUSIONS: Cell mitotic activity and coordinated cell death appear to be involved in growth and remodeling, which are activated by the mechanical input that occurs during bladder cycling. Fetal urinary diversion results in a repression of mitotic and apoptotic activity, limiting normal bladder growth and remodeling.

Neurons respond directly to mechanical deformation with pannexin-mediated ATP release and autostimulation of P2X7 receptors.

Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide (10)panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin-dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X(7) receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X(7) receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X(7) receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X(7) receptor, is neuroprotective for stretched neurons.

Beta and gamma-sarcoglycans are decreased in the detrusor smooth muscle cells of the partially obstructed rabbit bladder.

PURPOSE: We evaluated and quantified the levels of sarcoglycans present in the detrusor muscle layer of rabbits with partial bladder outlet obstruction. MATERIALS AND METHODS: Rabbits underwent surgery, as previously described, to partially obstruct the urethra. One, 3, 7 and 14 days after obstruction the detrusor muscle layer was dissected free of the remaining bladder tissue and extracted with detergent to isolate the transmembrane components of the dystroglycan-glycoprotein complex. Several components of the dystroglycan-glycoprotein complex were characterized and quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis several bands were noted on gels with a molecular weight (43 and 35 kDa, respectively) corresponding to beta and gamma-sarcoglycan. As obstruction progressed longitudinally, the levels of beta and gamma-sarcoglycan showed progressive decrease at the protein level with beta-sarcoglycan levels recovering at later time points. Bladders with a functional physiology that showed more advanced symptoms of dysfunction had a greater decrease in beta and gamma-sarcoglycan protein. CONCLUSIONS: The levels of beta and gamma-sarcoglycan progressively change with obstruction with greater changes occurring in the levels of gamma-sarcoglycan. It is likely that alterations in the dystroglycan-glycoprotein complex are responsible for some of the changes in muscle physiology that occur as a consequence of obstruction.

Altered extracellular matrix expression in the diverted fetal sheep bladder.

PURPOSE: It is unclear whether filling and emptying are important to bladder development. We tested this in an experimental preparation. MATERIAL AND METHODS: Urinary diversion was performed in 7 fetal lambs at 90 days of gestation and 6 unoperated fetal lambs served as controls. Transmural sections were analyzed for changes in tissue layer thickness and/or composition after 14 days of urinary diversion. Matrix mRNA levels (collagen I and III, and FN) as well as the cytokines/growth factors IGF-1, EGR-1, WT-1 and BCL-2 were quantified by real-time polymerase chain reaction. Hydroxyproline measurements of total collagen and collagen subtype quantification were done by enzyme-linked immunosorbent assay. RESULTS: Diverted fetal bladders showed a 27% and 57% decrease in mucosal and detrusor muscle layer thickness, respectively. In contrast, there was a 270% increase in serosal layer thickness in diverted bladders. The mRNA levels of COL1A1, COL3A1, IGF-1, EGR-1 and the anti-apoptotic gene BCL-2 were increased significantly in the serosal/detrusor layer of diverted bladders. In the mucosa levels of these mRNAs remained unchanged except for those of FN and WT-1, which were significantly decreased and increased, respectively. Total collagen, and type I and III collagen protein levels were significantly increased in diverted bladders. CONCLUSIONS: The lack of mechanical loading in diverted bladders leads to the arrest of detrusor smooth muscle growth, and concurrent fibrosis and thickening of the serosal layer. Changes in the levels of IGF-1, BCL-2 and EGR-1 likely have regulatory roles that affect the smooth muscle phenotype in the detrusor layer.

Smooth muscle trans-membrane sarcoglycan complex in partial bladder outlet obstruction.

The urinary bladder experiences both distension and contraction as a part of the normal filling and emptying cycle. To empty properly, tension generated intracellularly in a smooth muscle cell must be smoothly and efficiently transferred across its sarcolemma to the basement membrane, which mediates its binding to both the extracellular matrix and to other cells. As a consequence of urethral obstruction, the bladder cannot generate appropriate force to contract the organ, thereby leading to inefficient emptying and associated sequelae. In this study, an animal model of urethral obstruction was utilized to study the membrane-associated structures that transfer tension across the sarcolemma of bladder smooth muscle cells. Immunohistochemical localization of key components of the smooth muscle tension transfer apparatus (TTA) was performed utilizing specific antibodies against:(1) the alpha-chains of type IV collagen, a basement membrane component, and (2) beta-sarcoglycan, an integral membrane protein that is a participant in the physical linkage between the cytoskeleton and the basement membrane. We demonstrate, in obstructed animals, that there is a pronounced disruption of the TTA with a physical displacement of these two components that can be demonstrated at the level of the light microscope using scanning confocal microscopy. Electron microscopy further demonstrates significant increases in the size of the junctional plaques between smooth muscle cells.

Compression and tension: differential effects on matrix accumulation by periodontal ligament fibroblasts in vitro.

Human periodontal ligament fibroblasts were subjected to 10% cyclic equibiaxial tensional and compressive forces in vitro. Media supernatants were analyzed for changes in total protein, extracellular matrix proteins type I collagen and fibronectin, as well as MMP expression by gelatin zymography and Western blot. RNA analyses for changes in collagen, MMP-2, and TIMP-2 were carried out by either Real-time PCR and/or Northern blot. Application of compressional forces resulted in decreases in type I collagen and fibronectin protein, Col1A1 RNA, and increases in total protein, MMP-2 protein (latent and active), and MMP-2 RNA. TIMP-2 RNA was unchanged by compressive forces. In contrast, tensional forces increased total protein, type I collagen, Col1A1 RNA, as well as MMP-2 and TIMP-2 RNA. These studies show that cells can perceive two different forms of mechanical stimuli and respond in a differential manner relative to extracellular matrix synthesis and degradation.

Matrix synthesis by bladder smooth muscle cells is modulated by stretch frequency.

The bladder is a physically active organ that undergoes periodic stretching as a part of its normal function. To determine the role that stretching or mechanical deformation may play in altering the synthetic phenotype of bladder wall cells, a series of experiments were carried out to quantify several extracellular matrix (ECM) messenger ribonucleic acids (mRNAs) and their corresponding protein levels after mechanical challenge. Bladder smooth muscle cells were grown on distensible membranes in an apparatus that can reliably and reproducibly subject cells to well-characterized periods of mechanical stretching. For this study, cultured bovine bladder cells were subjected to cyclic mechanical deformation of varying frequencies to determine if this variable altered ECM expression. Using this experimental system, we demonstrated that smooth muscle cells were acutely sensitive to mechanical deformation and showed alteration in the synthesis of the major fibrillar collagens, types I and III. Concomitant analyses of mRNA in these cells show that levels of type I collagen correlate with mRNA levels at all frequencies except at 60 cycles/min, and, thus, type I production appears to be transcriptionally regulated. Interestingly, type III protein levels do not correlate with mRNA measurements except at 20 cycles/min, and, therefore, a different regulatory mechanism likely governs type III production. These studies demonstrate that smooth muscle cell ECM secretory phenotype can be altered by the frequency of mechanical deformation experienced by the cells. These data support the concept that stretching of the bladder wall affects the secretory phenotype of smooth muscle cells and can result in an altered ECM composition.

Regulation of Cyr61/CCN1 gene expression through RhoA GTPase and p38MAPK signaling pathways.

Cysteine-rich protein 61 (Cyr61/CCN1) is an angiogenic factor and a member of a family of growth factor-inducible immediate-early genes with functions in cell adhesion, proliferation and differentiation. We investigated the regulatory mechanisms and signaling pathways involved in Cyr61/CCN1gene activation in smooth muscle cells. Treatment of these cells with sphingosine 1-phosphate (S1P), a bioactive lysolipid, increased rapidly but transiently the expression of the Cyr61/CCN1 gene at both the mRNA and protein levels. Cyr61/CCN1 mRNA stability was not altered but the transcription rate of the Cyr61/CCN1 gene was increased fivefold in isolated nuclei from S1P-stimulated cells indicating that the level of control is primarily transcriptional. Transfection experiments showed that a 936-bp promoter fragment of the human Cyr61/CCN1 gene is functional and induces a reporter gene activity in S1P-treated cells. Using a combination of cis-element mutagenesis and expression of dominant negative inhibitors of transcription factors, we showed that both a CRE and AP-1 site and their cognate transcription factors, cAMP response element binding protein (CREB) and AP-1, were responsible for the promoter activity in S1P-stimulated cells. Furthermore, by using either pharmacological inhibitors or active forms of known signaling molecules, we showed that inducible Cyr61/CCN1 gene expression occurs through RhoA GTPase and that additional signaling through the p38 pathway is required. In particular, p38 seems to regulate Cyr61/CCN1 promoter activity through modulation of phosphorylation of CREB and the CREB kinase, MSK1. These findings demonstrate the transcriptional regulation of the Cyr61/CCN1 gene and provide clues to the signaling molecules and transcription factors involved in such regulation.