Fibroblast-myofibroblast Transformation Research Articles

Increased extracellular matrix stiffness regulates myofibroblast transformation through induction of autophagy-mediated Kindlin-2 cytoplasmic translocation

The extracellular matrix (ECM) mechanical properties regulate biological processes, such as fibroblast-myofibroblast transformation (FMT), which is a crucial component in pelvic organ prolapse (POP) development. The ‘Kindlin-2’ protein, expressed by fibroblasts, plays an important role in the development of the mesoderm, which is responsible for connective tissue formation; however, the role of Kindlin-2 in FMT remains to be explored. In this study, we aimed to explore the role of Kindlin-2 in FMT as it relates to POP. We found that ECM stiffness induces autophagy to translocate Kindlin-2 to the cytoplasm of L929 cells, where it interacts with and degrades MOB1, thereby facilitating Yes-associated protein (YAP) entry into the nucleus and influencing FMT progression. Stiffness-induced autophagy was inhibited when using an autophagy inhibitor, which blocked the translocation of Kindlin-2 to the cytoplasm and partially reversed high-stiffness-induced FMT. In patients with POP, we observed an increase in cytoplasmic Kindlin-2 and nuclear YAP levels. Similar changes in vaginal wall-associated proteins were observed in a mouse model of acute vaginal injury. In conclusion, Kindlin-2 is a key gene affecting ECM stiffness, which regulates FMT by inducing autophagy and may influence the development of POP.

Zeolitic imidazolate framework-90 loaded with methylprednisolone sodium succinate effectively reduces hypertrophic scar in vivo.

Hypertrophic scar (HS) is characterized by an abnormal fibroblast-myofibroblast transformation; non-apoptosis of fibroblasts; and redundant expression of TGF-β1, VEGF, α-SMA, and collagen I/III. An HS affects patients' physical and psychological quality of life, leading to joint dysfunction and skin cancer. However, there is currently no satisfactory drug to treat this disorder. In this study, we constructed methylprednisolone sodium succinate (MPSS) encapsulated ZIF-90 (MPSS@ZIF-90) for the effective treatment of an HS. The encapsulation of MPSS in ZIF-90 can achieve the controllable drug release of MPSS and prolong its effective treatment time. MPSS@ZIF-90 enhanced the apoptosis of human hypertrophic scar fibroblasts and downregulated the overexpression of TGF-β1, VEGF, α-SMA, and collagen I/III both in vitro and in vivo. The instant injection of MPSS@ZIF-90 effectively intervened with the formation of the HS after 28 days. On the contrary, MPSS@ZIF-90 greatly reduced the HS with two injections and 14 days of treatment after the HS was formed. This work provides evidence of effective intervention in the formation of an HS and the therapeutic effectiveness of MPSS@ZIF-90 with short treatment periods in vivo. It suggests that MPSS@ZIF-90 can be used as a biomedical option in the treatment of skin wounds and may reveal the potential molecular basis for promising future antifibrotic agents against scarring.

Noscapine alleviates unilateral ureteral obstruction-induced inflammation and fibrosis by regulating the TGFβ1/Smads signaling pathways

Renal fibrosis is a common pathway leading to progressive renal function loss in various forms of chronic kidney disease. Many fibrogenic factors regulate renal fibrosis; two key players are post-injury inflammation and transforming growth factor-β1 (TGF-β1)-induced myofibroblast differentiation. Myofibroblast differentiation is tightly regulated by the microtubule polymerization. Noscapine, an antitussive plant alkaloid, is a potent microtubule-interfering agent previously identified as a potential anticancer compound. Here, we examined how noscapine affects renal fibrogenesis in an in vitro renal fibroblast model and an in vivo unilateral ureteral obstruction (UUO) model. UUO mice were intraperitoneally treated with noscapine at 1 day before UUO surgery and daily thereafter. At 7 days post-surgery, kidneys were collected for further analysis. To analyze whether noscapine inhibits downstream TGF-β1-related signaling, we pre-incubated NRK-49F fibroblasts with noscapine and then performed TGF-β1 stimulation. In UUO mice, noscapine attenuated extracellular matrix protein deposition and the expression levels of type I collagen, type IV collagen, α-smooth muscle actin, and fibronectin. In addition, noscapine decreased tubulointerstitial inflammation in UUO kidneys by reducing TLR2 expression, modulating NLRP3 inflammasome activation, reducing macrophage infiltration, and antagonizing the M2 macrophage phenotype. Furthermore, noscapine pre-incubation suppressed the TGF-β1-induced fibroblast-myofibroblast transformation by downregulating the TGF-β/Smads signaling pathways in NRK-49F cells. These results suggest that noscapine reduces tubulointerstitial inflammation and fibrosis in the kidneys of UUO mice and inhibits the fibroblast-myofibroblast transformation induced by TGF-β1. Noscapine is an over-the-counter antitussive that has been used safely for several decades. Therefore, noscapine is an attractive therapeutic agent for inhibiting renal tubulointerstitial fibrosis.

Inhibition of miR-543 alleviates cardiac fibroblast-to-myofibroblast transformation and collagen expression in insulin resistance via targeting PTEN

BackgroundMyocardial interstitial fibrosis is an important manifestation of diabetic heart disease, and insulin resistance is one of the mechanisms of myocardial interstitial fibrosis. Some studies have found that miR-543 is associated with insulin resistance, but whether it plays a role in diabetic myocardial interstitial fibrosis remains unclear. This study aimed to investigate the role of miR-543 in diabetic myocardial interstitial fibrosis. MethodsThe combination of high glucose and high insulin was used to establish an insulin-resistant myocardial fibroblast model. The expression levels of miR-543, α-SMA, collagen Ⅰ, collagen Ⅲ and PTEN were detected. Cell proliferation and migration were detected. Luciferase reporter gene assay was used to verify the targeting relationship between miR-543 and PTEN. ResultsThe expression of miR-543 was up-regulated in myocardial fibroblasts with insulin resistance, which was consistent with the results of bioinformatics analysis. The proliferation and migration levels of myocardial fibroblasts in insulin-resistant states were increased, and the expression levels of α-SMA, collagen Ⅰ and collagen Ⅲ were also increased. Inhibition of miR-543 expression could reverse the above changes. Target gene prediction and dual luciferase reporter assay demonstrated that miR-543 could bind to the 3′UTR region of PTEN. Moreover, the effect of miR-543 on insulin-resistant myocardial fibroblasts is mediated by targeting PTEN. ConclusionsInhibition of miR‐543 can reduce myocardial fibroblast-myofibroblast transformation and collagen expression in insulin-resistant states by targeting PTEN.

Celastrol Targets Cullin-Associated and Neddylation-Dissociated 1 to Prevent Fibroblast-Myofibroblast Transformation against Pulmonary Fibrosis.

Celastrol (CEL), a pentacyclic triterpene compound, has been proven to have a definite antipulmonary fibrosis effect. However, its direct targets for antipulmonary fibrosis remain unknown. In this study, we designed and synthesized a series of celastrol-based probes to identify the direct targets in human pulmonary fibroblasts using an activity-based protein profiling strategy. Among many fished targets, we identified a key protein, cullin-associated and neddylation-dissociated 1 (CAND1), which was involved in fibroblast-myofibroblast transformation (FMT). More importantly, we found that the inhibitory effect of celastrol on FMT is dependent on CAND1, through improving the interactions between CAND1 and Cullin1 to promote the activity of Skp1/Cullin1/F-box ubiquitin ligases. In silico studies and cysteine mutation experiments further demonstrated that Cys264 of CAND1 is the site for conjugation of celastrol. This reveals a new mechanism of celastrol against pulmonary fibrosis and may provide a novel therapeutic option for antipulmonary fibrosis.

Activation of adenosine receptors improved cardiac dysfunction induced by myocardial infarction

Abstract Introduction Acute myocardial infarction (MI) is characterized by an intense inflammatory response leading to cardiac remodeling and subsequent cardiac dysfunction. Purpose Activation of adenosine A2A and A3 receptors induced by LASSBio-1027 could improve cardiac function, through the reduction of the inflammatory response and the activated cardiac fibroblasts (CF). Methods The Animal Care and Use Committee approved the experimental protocols. MI was induced in male Wistar rats by ligation of the anterior descending coronary artery and confirmed by transthoracic echocardiography. The experimental groups were Sham and MI treated orally with either vehicle or LASSBio-1027 (30 or 70 μmol/kg) during 7 days. At the endpoint, hemodynamic parameters were evaluated and hearts were prepared for histological and immunohistochemical analysis for fibrosis and cellular infiltrate detection and for α-SMA, TNF-α, iNOs, c-fos, p38MAPK expression, respectively. Production of reactive oxygen species and collagen in CF with H2O2–induced oxidative stress was observed in the presence or absence of LASSBio-1027 (10 μM). Results Diastolic dysfunction was detected in MI group because the filling pressure (E/e') and the left ventricular end diastolic pressure (LVEDP) were increased from 22.9±1.6 to 37.0±3.7 and from 3.2±0.9 to 18.2±2.2 mmHg (p<0.01), respectively. LASSBio-1027 (70 μmol/kg) prevented diastolic dysfunction since E/e' and LVEDP reduced to 23.8±5.3 and 6.4±1.6 mmHg (p<0.01). Adenosine receptor agonist improved systolic dysfunction in MI group with increase of ejection fraction from of 30.1±3.1 to 48.4±4.8% (p<0.05). As shown in figure 1, MI promoted an increase in collagen deposition, cellular infiltrate, expression of SMA-α, TNF-α, iNOs, c-fos and p38MAPK which confirmed the intense fibrosis, cell proliferation and inflammation. Oral administration of LASSBio-1027 reduced those parameters in a dose dependent manner. LASSBio-1027 reduced H2O2-induced fibroblast-myofibroblast transformation with the decrease of stain density of α-SMA from 1794.4 to 574.4 staining area/cell and collagen production from 33.7±9.6 to 22.1±18.9% (p<0.05). Effects of LASSBio-1027 were inhibited by the antagonists of A2A (ZM-241385) and A3 (MRE3008F20) receptors. Conclusions Activation of adenosine A2A and A3 receptors by LASSBio-1027 improved cardiac function and remodeling induced by MI. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): CNPq. CAPES, FAPERJ Histological AnalysisImmunohistochemistry analysis

Resveratrol Ameliorates Cardiac Remodeling in a Murine Model of Heart Failure With Preserved Ejection Fraction

Objective: Accumulating evidence suggested that resveratrol (RES) could protect against adverse cardiac remodeling induced by several cardiovascular diseases. However, the role of RES in the setting of heart failure with preserved ejection fraction (HFpEF) and the underlying mechanisms of its action remain understood. This study was to determine whether RES could ameliorate HFpEF-induced cardiac remodeling and its mechanisms. Methods: In vivo, C57BL/6 mice served as either the sham or the HFpEF model. The HFpEF mice model was induced by uninephrectomy surgery and d-aldosterone infusion. RES (10 mg/kg/day, ig) or saline was administered to the mice for four weeks. In vitro, transforming growth factor β1 (TGF-β1) was used to stimulate neonatal rat cardiac fibroblasts (CFs) and Ex-527 was used to inhibit sirtuin 1 (Sirt1) in CFs. Echocardiography, hemodynamics, western blotting, quantitative real-time PCR, histological analysis, immunofluorescence, and ELISA kits were used to evaluate cardiac remodeling induced by HFpEF. Sirt1 and Smad3 expressions were measured to explore the underlying mechanisms of RES. Results: HFpEF mice developed left ventricular hypertrophy, preserved ejection fraction, diastolic dysfunction, and pulmonary congestion. Moreover, HFpEF mice showed increased infiltration of neutrophils and macrophages into the heart, including increased interleukin (IL)-1β, IL-6, and TNF-α. We also observed elevated M1 macrophages and decreased M2 macrophages, which were exhibited by increased mRNA expression of M1 markers (iNOS, CD86, and CD80) and decreased mRNA expression of M2 markers (Arg1, CD163, and CD206) in HFpEF hearts. Moreover, HFpEF hearts showed increased levels of intracellular reactive oxygen species (ROS). Importantly, HFpEF mice depicted increased collagen-I and -III and TGF-β mRNA expressions and decreased protein expression of phosphorylated endothelial nitric-oxide synthase (p-eNOS). Results of western blot revealed that the activated TGF-β/Smad3 signaling pathway mediated HFpEF-induced cardiac remodeling. As expected, this HFpEF-induced cardiac remodeling was reversed when treated with RES. RES significantly decreased Smad3 acetylation and inhibited Smad3 transcriptional activity induced by HFpEF via activating Sirt1. Inhibited Sirt1 with Ex-527 increased Smad3 acetylation, enhanced Smad3 transcriptional activity, and offset the protective effect of RES on TGF-β–induced cardiac fibroblast–myofibroblast transformation in CFs. Conclusion: Our results suggested that RES exerts a protective action against HFpEF-induced adverse cardiac remodeling by decreasing Smad3 acetylation and transcriptional activity via activating Sirt1. RES is expected to be a novel therapy option for HFpEF patients.

2,5-Dimethylcelecoxib attenuates cardiac remodeling after cryoinjury-induced myocardial infarction by suppressing the fibroblast-myofibroblast transformation

Secretion of extracellular matrix by myofibroblasts, transform from fibroblasts and express alpha smooth muscle actin (aSMA), lead to cardiac fibrosis and progress cardiac remodeling. We previously reported the 2,5-dimethylcelecoxib (DM-C) attenuated cardiac remodeling in different type of cardiac hypertrophy models. However, as the effect of DM-C on myofibroblasts transformation and cardiac remodeling after myocardial infarction (MI) are still unclear, we tried to clarify these points. In in vitro study, dermal fibroblasts from SD rat were used for analysis of myofibroblast transformation. In in vivo study, cryoinjury-induced MI (CMI) mouse model was used. In vitro studies showed that DM-C significantly suppressed the expression of aSMA and the phosphorylation levels of Akt, GSK-3 b and Smad2/3, all of them were elevated by TGF-b treatment. In vivo studies showed that the expression of aSMA and fibrosis area were decreased in DM-C group compared with control group. Further, evaluation of the cardiac function revealed that DM-C-treatment alleviated the reduction of the ejection fraction. These results suggest that DM-C attenuates cardiac remodeling after CMI by inhibiting fibrosis via suppression of the myofibroblast transformation. Therefore, DM-C has potential as the novel drug for treatment of cardiac remodeling after MI.

Irisin attenuates angiotensin II-induced cardiac fibrosis via Nrf2 mediated inhibition of ROS/ TGFβ1/Smad2/3 signaling axis

Angiotensin II-related cardiac fibrosis is one of the key pathological changes of the hypertrophied left ventricle in various heart disease. Irisin was recently reported to confer cardio-protective and anti-oxidative effects, while whether it can reverse the renin–angiotensin–aldosterone system(RAAS) activation related(angiotensin II-induced) cardiac fibrosis is unknown. In this study, we found that angiotensin II-induced cardiac dysfunction and fibrotic responses were dampened by irisin treatment in mice. Mechanistically, angiotensin II induced robust ROS generation, which in turn triggered activation of pro-fibrotic TGFβ1-Smad2/3 signaling and subsequent collagen synthesis and fibroblast-myofibroblast transformation in cardiac fibroblasts. In contrast, Irisin treatment suppressed angiotensin II-induced ROS generation, TGFβ1 activation, collagen synthesis and fibroblast-myofibroblast transformation, the effects of which was accompanied by Nrf2 activation and also abolished by a Nrf2 targeted siRNA. Taken together, we here identified irisin as a promising anti-fibrotic therapeutic for angiotensin II-related cardiac fibrosis.

Antifibrotic effect of mitomycin-C on human vocal cord fibroblasts.

Acquired laryngotracheal stenosis is a potentially life-threatening situation and a very difficult and challenging problem in laryngology. Therefore, new trends and innovative approaches based on antifibrotic drugs and minimally invasive regimens are being developed to attenuate laryngotracheal fibrosis and scarring. The purpose of this study was to examine the efficacy of mitomycin-C (MMC) to reverse the transforming growth factor (TGF)-β-induced differentiation of MRC-5 fibroblast and human primary vocal cord fibroblasts to reveal the possible applicability of MMC to laryngotracheal fibrotic conditions. Human primary fibroblast cells were isolated from vocal cord specimens of patients undergoing total laryngectomy. The established primary vocal cord fibroblast cell cultures as well as the MRC-5 human fibroblast cells were treated with 5 ng/mL TGF-β alone and then with 0.5 µg/mL MMC for 24 hours. Differentiation of fibroblasts was characterized by α-smooth muscle actin (α-SMA) immunhistochemistry, Western blot analysis, and real-time polymerase chain reaction. Cell motility was assessed by wound-healing assay. Elevated α-SMA mRNA and protein expression as well as increased cell motility were observed upon TGF-β exposures. However, after MMC treatments the TGF-β-induced fibroblasts exhibited a significant decrease in α-SMA expression and wound-healing activity. Therefore, TGF-β-stimulated fibroblast-myofibroblast transformation was reversed at least in part by MMC treatment. Histopathological examinations of tissue specimens of a laryngotracheal stenosis patient supported these findings. Antifibrotic effects of MMC were demonstrated on the human MRC-5 cell line and on primary vocal cord fibroblast cultures. These results verify that MMC can be used with success to reverse upper airway stenosis by reverting the myofibroblast phenotype. NA Laryngoscope, 129:E255-E262, 2019.

Long noncoding RNA TUG1 promotes cardiac fibroblast transformation to myofibroblasts via miR‑29c in chronic hypoxia.

Cardiac fibroblast‑myofibroblast transformation(FMT) contributes to the fibrotic deterioration evoked by chronic hypoxia. Growing evidence implicates long noncoding RNAs (lncRNAs) in various types of cardiac physiological and pathological processes, especially in cardiac fibrosis. In the present study, the lncRNA Taurine Upregulated Gene1(TUG1), reported as a regulator of hypoxia fibrosis in the lungs, was found to also be an important regulator of cardiac FMT. Specifically, the possible role of TUG1 in cardiac FMT and fibrosis under chronic hypoxia was investigated. It was revealed that the degree of fibrosis in heart tissues collected from congenital heart surgery patients with low pulse oxygen saturation and mice housed under chronic hypoxic and atmospheric pressure conditions was negatively correlated with pulse oxygen saturation. Moreover, TUG1 expression was positively correlated with the degree of fibrosis but negatively correlated with pulse oxygen saturation. Cardiac fibroblasts showed increased myofibroblast marker, collagen I and α‑SMA expression levels as the hypoxia time increased. TUG1 knockdown ameliorated the hypoxia‑induced FMT. A bioinformatics analysis predicted that TUG1 had miR‑29c binding sites in its 3'‑UTR and miR‑29c is a key regulator of cardiac fibrosis. The present study demonstrated that TUG1, along with miR‑29c, may contribute to cardiac FMT activation and promote fibrosis in chronic hypoxia.

Loss of IκB kinase β promotes myofibroblast transformation and senescence through activation of the ROS-TGFβ autocrine loop.

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0241-6) contains supplementary material, which is available to authorized users.

Role of Notch signaling in the mammalian heart

Notch signaling is an evolutionarily ancient, highly conserved pathway important for deciding cell fate, cellular development, differentiation, proliferation, apoptosis, adhesion, and epithelial-to-mesenchymal transition. Notch signaling is also critical in mammalian cardiogenesis, as mutations in this signaling pathway are linked to human congenital heart disease. Furthermore, Notch signaling can repair myocardial injury by promoting myocardial regeneration, protecting ischemic myocardium, inducing angiogenesis, and negatively regulating cardiac fibroblast-myofibroblast transformation. This review provides an update on the known roles of Notch signaling in the mammalian heart. The goal is to assist in developing strategies to influence Notch signaling and optimize myocardial injury repair.

Notch Signaling May Negatively Regulate Neonatal Rat Cardiac Fibroblast-Myofibroblast Transformation

Cardiac fibroblast-myofibroblast transformation (CMT) is a critical event in the initiation of myocardial fibrosis. Notch signaling has been shown to regulate myofibroblast transformation from other kinds of cells. However, whether Notch signaling is also involved in CMT remains unclear. In the present study, expressions of Notch receptors in cardiac fibroblasts (CFs) were examined, effects of Notch signaling inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) and transforming growth factor-beta1 (TGF-beta1) on CMT were determined by increasing alpha-smooth muscle actin (alpha-SMA) expression and collagen synthesis, and Notch signaling was examined by analyzing expressions of Notch receptors. The results showed that: (1) Notch receptor 1, 2, 3 and 4 were all expressed in CFs; (2) DAPT promoted CMT in a time-dependent manner; (3) During the period of CMT induced by TGF-beta1, expressions of Notch receptor 1, 3 and 4 in CFs were down-regulated, whereas there was no change for Notch receptor 2. Moreover, the downtrends of Notch 1, 3 and 4 were corresponding to the trend growth of alpha-SMA expression and collagen synthesis. These results suggested that inhibiting of Notch signaling might promote CMT. The down-regulations of Notch receptor 1, 3 and 4 induced by TGF-beta1 may facilitate CMT. In conclusion, inhibition of Notch signaling might be a novel mechanism of CMT in myocardial fibrosis.

The use of light‐emitting diode therapy in the treatment of photoaged skin

Light-emitting diode (LED) therapy is an increasingly popular methodology for the treatment of sun damage. Combination use of light wavelengths reported to stimulate collagen synthesis and accelerate fibroblast-myofibroblast transformation may display a composite rejuvenative effect. To clinically assess reduction in sun damage signs following a 5-week course of LED therapy and to assess subject's perception of the treatment. Thirteen subjects with wrinkles or fine lines in the periorbital and nasolabial region and those presenting Glogau scale photodamage grade II-III received nine 20-min duration light treatments using the Omnilux LED system. The treatments combined wavelengths of 633 and 830 nm at fluences of 126 and 66 J/cm(2), respectively. Sun-damage reduction was assessed at 6, 9, and 12 weeks by clinical photography and patient satisfaction scores. The majority of subjects displayed "moderate" (50%) or "slight" (25%) response to treatment at investigator assessment. Treatment of the periorbital region was reported more effective than the nasolabial region. At 12-week follow-up, 91% of subjects reported improved skin tone, and 82% reported enhanced smoothness of skin in the treatment area. Good response to LED therapy has been shown in this modest sample. Larger trials are needed to assess optimum frequency of light treatments and overall treatment time.

Focal Adhesions in (Myo)fibroblasts Scaffold Adenylyl Cyclase with Phosphorylated Caveolin

Fibroblast-myofibroblast transformation, a critical event for enhanced extracellular matrix deposition, involves formation of an actin stress fiber contractile apparatus that radiates from focal adhesions (FA) in the plasma membrane. Activation of adenylyl cyclase (AC, i.e. increases in cAMP) negatively regulates such transformation. Caveolae and their resident protein caveolins scaffold signaling molecules, including AC isoforms, whereas phosphorylated caveolin-1 (phospho-cav-1) may localize at FA. Here, we used adult rat cardiac fibroblasts to examine distribution and expression of AC, phospho-cav-1, and FA proteins to define mechanisms that link increases in cAMP to caveolin-1 phosphorylation, actin/FA assembly, and fibroblast-myofibroblast transformation. Sucrose density gradient centrifugation, immunoblot, and immunohistochemical analysis revealed that, unlike cav-1, phospho-cav-1 enriches in membrane fractions that express FA proteins and localize at the ends of actin stress fibers. We detected AC in both cav-1 and phospho-cav-1 immunoprecipitates, but FA kinase (FAK), phospho-FAK (FAK Tyr-397), paxillin, and vinculin were detected only in phospho-cav-1 immunoprecipitates. Treatment with the AC activator forskolin or a cAMP analog increased cav-1 phosphorylation but decreased FAK Tyr-397 phosphorylation in a cAMP-dependent protein kinase-dependent manner. These events preceded actin cytoskeletal disruption, an effect that was blocked by small interfering RNA knock-down of cav-1. Inhibition of protein tyrosine phosphatase 1B abrogated cAMP-mediated disruption of actin cytoskeleton, cav-1 phosphorylation, and FAK Tyr-397 dephosphorylation. The data thus define a novel organization of signaling molecules that regulate fibroblasts: scaffolding of AC by phospho-cav-1 at FA sites in a caveolae-free microdomain along with components that mediate inhibition of actin/FA assembly and fibroblast-myofibroblast transformation via increases in cAMP.