Myeloid differential protein-2 inhibition improves diabetic cardiomyopathy via p38MAPK inhibition and AMPK pathway activation

Myeloid differential protein-2 (MD2) has been shown to play a critical role in the progression of diabetic cardiomyopathy (DCM). This study aims to explore the non-inflammatory mechanisms mediated by MD2 in DCM and to test the therapeutic effects of MD2 inhibitor C30 on DCM. Streptozotocin (STZ) was used to construct DCM model in wild-type and MD2 knockout mice. The collected heart samples were subjected to RNA-sequencing assay. Gene set enrichment analysis of the RNA-seq data indicated that MD2 knockout was associated with energy metabolism pathways in diabetic mouse heart. Further data showed that AMPK pathway was impaired under high glucose condition, which was mediated by p38MAPK activation. MD2 knockout or pharmacological inhibitor C30 completely rescued AMPK signaling through p38MAPK inhibition. Importantly, C30 treatment significantly prevented myocardial damage and dysfunction in T1DM mice evidenced by improved cardiac function and reduced cardiomyocyte apoptosis and cardiac fibrosis. Furthermore, the therapeutic effect of C30 on DCM was correlated to p38MAPK inhibition and AMPK pathway activation in vivo and in vitro. In conclusion, MD2 inhibition exhibits therapeutic effects on DCM through p38MAPK inhibition and AMPK activation, which enables MD2 a promising target for DCM treatment by suppressing metaflammation and improving cardiac metabolism.     

Leptin regulates MMP-2, TIMP-1 and collagen synthesis via p38 MAPK in HL-1 murine cardiomyocytes

A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observed that leptin enhanced collagen synthesis determined by both proline incorporation and picrosirius red staining of conditioned media. Pro-collagen type-I and pro-collagen type-III expression, measured by real-time PCR and Western blotting were also increased by leptin, effects which were again attenuated by SB203580. In summary, these results demonstrate the potential for leptin to play a role in mediating myocardial ECM remodeling and that the p38 MAPK pathway plays an important role in mediating these effects.     

Mechanical stretch promotes matrix metalloproteinase-2 and prolyl-4-hydroxylase α1 production in human aortic smooth muscle cells via Akt-p38 MAPK-JNK signaling

Hypertension can increase mechanical stretch on the vessel wall, an important stimulus that induces collagen remodeling. Prolyl-4-hydroxylaseα1 (P4Hα1) and matrix metalloproteinases (MMPs) are essential for collagen synthesis and degradation. However, the effect of mechanical strain and collagen synthesis remains largely unknown. This study aimed to identify the effect of stretch on MMPs and P4Hα1 and the involved signaling pathways. Human aortic smooth muscle cells (HASMCs) were stimulated with mechanical stretch (0, 10% and 18% strain), and production of P4Hα1 as well as production and gelatinolytic activity of MMP-2 was force-dependently increased. Mechanical stretch at 18% also increased the expression of type I and III collagen and the phosphorylation of Akt, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). MMP-2 production and activity enhanced by 18% stretch were inhibited by the PI3K/Akt inhibitor LY294002. Blockade of p38 MAPK or JNK inhibited the promoting effect of stretch on P4Hα1. The in vivo model of aortic banding showed increased protein levels of MMP-2, P4Hα1 and collagen I and III in the aorta. Thus, mechanical stretch increased MMP-2 and P4Hα1 expression in HASMCs via AKT-P38 MAPK-JNK signaling, thereby inducing vascular remodeling.     

Role of Smad2/3 and p38 MAP kinase in TGF-β1-induced epithelial-mesenchymal transition of pulmonary epithelial cells

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-β1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-β1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-β1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-β1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-β1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-β1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.     

Peroxisome proliferator-activated receptor-γ ligands attenuate brain natriuretic peptide production and affect remodeling in cardiac fibroblasts in reoxygenation after hypoxia

Cardiac fibroblasts (CFs) participate in cardiac remodeling after hypoxic cardiac damage, and remodeling is thought to be mediated by CF synthesis of brain natriuretic peptide (BNP). It is unknown whether the peroxisome proliferator-activated receptors (PPARs), which mediate cellular signaling for growth and migration, affect BNP synthesis and whether PPARs participate in regulation of extracellular matrix protein (ECM) expression for remodeling. We examined the production of BNP in cultured neonatal ventricular CFs and its signaling system on collagen synthesis and on activation of matrix metalloproteinases (MMPs) in reoxygenation after hypoxia. BNP mRNA was detected in CFs, and a specific BNP protein, BNP1-32, was secreted into the media. Abundance of collagen I and III was increased in the media at reoxygenation. mRNA and protein levels for MMP-2 and the tissue inhibitor of metalloproteinase (TIMP)-1 were enhanced in CFs at reoxygenation. These observations also were noted in CFs after incubation with angiotensin II (10 μM) for 24 h. Pretreatment with pioglitaozone (0.1–10 μM) attenuated BNP mRNA and protein abundance of collagen III, MMP-2, and TIMP-1 in CFs at reoxygenation. The secreted BNP was also decreased by pioglitaozone in the media. Furthermore, PPAR activators inhibited reoxygenation-induced activation of nuclear factor (NF)-kB. These results demonstrate that PPAR activators inhibit BNP synthesis in CFs and imply that PPAR activators may regulate ECM remodeling partially through the NF-kB-mediated pathway.     

Cellular localization of integrin isoforms in phenylephrine-induced hypertrophic cardiac myocytes

Cardiac hypertrophy is characterized by remodeling of the extracellular matrix (ECM). Integrins are cell-surface molecules that link the ECM to the cellular cytoskeleton where they play roles as signaling molecules and transducers of mechanical force. To clarify the possible roles of integrins in cardiac myocyte hypertrophy, we investigated the cellular localization and expression of ECM proteins and integrins in both normal cardiac myocytes and phenylephrine-induced hypertrophic myocytes. Addition of phenylephrine (PE) to cultured neonatal cardiac myocytes induced sarcomeric organization, increase in cell size, and synthesis of the hypertrophic marker, atrial natriuretic factor (ANF). In particular, fibronectin and collagen underwent dramatic localization changes during PE-induced cardiac hypertrophy. Significant changes were noted in the cellular localization of the respective collagen and fibronectin receptors, integrin alpha1 and alpha5, from diffuse to a sarcomeric banding pattern. Expression levels of integrins were also increased during hypertrophy. Treatment with okadaic acid (OA), an inhibitor of protein phosphatase 2A (PP2A), resulted in inhibition of hypertrophic response. These results suggest that dephosphorylation of integrin beta1 may be important in the induction of cardiac hypertrophy.     

Inhibition of JNK Aggravates the Recovery of Rat Hearts after Global Ischemia: The Role of Mitochondrial JNK

c-Jun N-terminal kinase (JNK), a stress-activated MAPK, is activated during cardiac ischemia-reperfusion (IR). The role of JNK inhibitors in cardioprotection against IR still remains controversial, in part, due to spill-over effects of non-specific inhibitors. In the present study, we sought to examine whether inhibition of JNK by SU3327, a specific JNK inhibitor that inhibits upstream JNK signaling rather than the kinase activity of JNK, improves cardiac function and reduces heart damage during IR. Hearts of male Sprague-Dawley rats perfused by Langendorff were subjected to 25 min of global ischemia followed by 30 min reperfusion in the presence or absence of SU3327. Cardiac function was monitored throughout the perfusion period. Myocardial damage was extrapolated from LDH activity in the coronary effluent. At the end of reperfusion, mitochondria were isolated and used to measure respiration rates and mitochondrial permeability transition pore opening. Protein analysis of mitochondria predictably revealed that SU3327 inhibited JNK phosphorylation. Although SU3327 significantly reduced cell damage during the first minutes of reperfusion, it did not improve cardiac function and, furthermore, reduced the mitochondrial respiratory control index. Interestingly, SU3327 activated the other stress-related MAPK, p38, and greatly increased its translocation to mitochondria. Mitochondrial P-JNK and P-p38 were co-immunoprecipitated with complex III of the electron transfer chain. Thus, JNK plays an essential role in cardiac signaling under both physiological and pathological conditions. Its inhibition by SU3327 during IR aggravates cardiac function. The detrimental effects of JNK inhibition are associated with reciprocal p38 activation and mitochondrial dysfunction.     

Regulators of cardiac fibroblast cell state

Fibroblasts are the primary regulator of cardiac extracellular matrix (ECM). In response to disease stimuli cardiac fibroblasts undergo cell state transitions to a myofibroblast phenotype, which underlies the fibrotic response in the heart and other organs. Identifying regulators of fibroblast state transitions would inform which pathways could be therapeutically modulated to tactically control maladaptive extracellular matrix remodeling. Indeed, a deeper understanding of fibroblast cell state and plasticity is necessary for controlling its fate for therapeutic benefit. p38 mitogen activated protein kinase (MAPK), which is part of the noncanonical transforming growth factor β (TGFβ) pathway, is a central regulator of fibroblast to myofibroblast cell state transitions that is activated by chemical and mechanical stress signals. Fibroblast intrinsic signaling, local and global cardiac mechanics, and multicellular interactions individually and synergistically impact these state transitions and hence the ECM, which will be reviewed here in the context of cardiac fibrosis.     

p38 MAPK Signaling in Postnatal Tendon Growth and Remodeling

Tendon is a dynamic tissue whose structure and function is influenced by mechanical loading, but little is known about the fundamental mechanisms that regulate tendon growth and remodeling in vivo. Data from cultured tendon fibroblasts indicated that the p38 MAPK pathway plays an important role in tendon fibroblast proliferation and collagen synthesis in vitro. To gain greater insight into the mechanisms of tendon growth, and explore the role of p38 MAPK signaling in this process, we tested the hypotheses that inducing plantaris tendon growth through the ablation of the synergist Achilles tendon would result in rapid expansion of a neotendon matrix surrounding the original tendon, and that treatment with the p38 MAPK inhibitor SB203580 would prevent this growth. Rats were treated with vehicle or SB203580, and subjected to synergist ablation by bilateral tenectomy of the Achilles tendon. Changes in histological and biochemical properties of plantaris tendons were analyzed 3, 7, or 28 days after overload, and comparisons were made to non-overloaded animals. By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon. The expansion in CSA initially occurred through the synthesis of a hyaluronic acid rich matrix that was progressively replaced with mature collagen. Pericytes were present in areas of active tendon growth, but never in the original tendon ECM. Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth. The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.     

Alpha2beta1 integrin promotes T cell survival and migration through the concomitant activation of ERK/Mcl-1 and p38 MAPK pathways

Integrin-mediated attachment to extracellular matrix (ECM) is crucial for cancer progression. Malignant T cells such as acute lymphoblastic leukemia (T-ALL) express β1 integrins, which mediate their interactions with ECM. However, the role of these interactions in T-ALL malignancy is still poorly explored. In the present study, we investigated the effect of collagen; an abundant ECM, on T-ALL survival and migration. We found that collagen through α2β1 integrin promotes the survival of T-ALL cell lines in the absence of growth factors. T-ALL cell survival by collagen is associated with reduced caspase activation and maintenance of Mcl-1 levels. Collagen activated both ERK and p38 MAPKs but only MAPK/ERK was required for collagen-induced T-ALL survival. However, we found that α2β1 integrin promoted T-ALL migration via both ERK and p38. Together these data indicate that α2β1 integrin signaling can represent an important signaling pathway in T-ALL pathogenesis and suggest that its blockade could be beneficial in T-ALL treatment.     

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403–410, 1998. © 1998 Wiley-Liss, Inc.     

Early induction of matrix metalloproteinase-9 transduces signaling in human heart end stage failure

Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic-MMP-2 and -9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP-2 and -9 in ailing to failing myocardium. Activation of MMP-2 and -9 leads to induction of proteinase activated receptor-1 (PAR-1). We hypothesize that the early induction of MMP-9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase-1 (ERK-1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP-2 and -9 gelatinolytic activities was made by real-time quantitative zymography. Gel phosphorylation staining for PAR-1 showed a significant increase in ICM hearts. Western blot and RT-PCR analysis and in-situ labeling, showed significant increased expression of PAR-1, ERK-1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP-9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end-stage human heart failure.     

Transdifferentiation-dependent expression of alpha-SMA in hepatic stellate cells does not involve TGF-beta pathways leading to coinduction of collagen type I and thrombospondin-2.

Hepatic stellate cells (HSC) cultured on plastic spontaneously transdifferentiate to a myofibroblast-like cell type (MFB). This model system of hepatic fibrogenesis is characterized by phenotypic changes of the cells and increased matrix synthesis. Here, we analyzed if transdifferentiation-dependent induction of ECM components, e.g., collagen type I and thrombospondin-2 (TSP-2), and phenotypic changes are coregulated events and if both processes are mediated via TGF-beta pathway(s). Blocking the TGF-beta-dependent p38 MAPK pathway in HSC with the specific inhibitor SB203580 strongly reduces collagen I and TSP-2 mRNA expression without inhibiting upregulation of the typical MFB-marker, alpha-smooth-muscle actin (alpha-SMA). Similarly, interference with the Smad2/3/4 pathway using dexamethasone also heavily decreased expression of collagen type I and TSP-2 whereas transdifferentiation of HSC to the typical morphology of MFB with loss of fat droplets and increasing alpha-SMA was unchanged. Further, p38 MAPK mediated induction of collagen I and TSP-2 expression by TGF-beta1 was still achieved in the presence of dexamethasone, showing that dexamethasone does not block p38 while it delays Smad2 phosphorylation and antagonizes stimulation of a Smad3/Smad4 dependent TGF-beta reporter construct. Interestingly, in contrast to SB203580 and dexamethasone, overexpression of the TGF-beta antagonist Smad7 reduced ECM expression and simultaneously inhibited morphologic transdifferentiation, indicating that Smad7 fulfills additional features in HSC. In conclusion, our data show that phenotypic changes of transdifferentiating HSC and induction of matrix synthesis are independent processes, the latter being stimulated by both, Smad dependent and MAPK dependent TGF-beta signaling.     

Adenosine A2A receptor signaling regulation of cardiac NADPH oxidase activity

Cardiac tissues express constitutively an NADPH oxidase, which generates reactive oxygen species (ROS) and is involved in redox signaling. Myocardial metabolism generates abundant adenosine, which binds to its receptors and plays important roles in cardiac function. The adenosine A2A receptor (A2AR) has been found to be expressed in cardiac myocytes and coronary endothelial cells. However, the role of the A2AR in the regulation of cardiac ROS production remains unknown. We found that knockout of A2AR significantly decreased (39+/-8%) NADPH-dependent O2- production in mouse hearts compared to age (10 weeks)-matched wild-type controls. This was accompanied by a significant decrease in Nox2 (a catalytic subunit of NADPH oxidase) protein expression, and down-regulation of ERK1/2, p38MAPK, and JNK phosphorylation (all P<0.05). In wild-type mice, intraperitoneal injection of the selective A2AR antagonist SCH58261 (3-10 mg/kg body weight for 90 min) inhibited phosphorylation of p47phox (a regulatory subunit of Nox2), which was accompanied by a down-regulated cardiac ROS production (48+/-8%), and decreased JNK and ERK1/2 activation by 54+/-28% (all P<0.05). In conclusion, A2AR through MAPK signaling regulates p47phox phosphorylation and cardiac ROS production by NADPH oxidase. Modulation of A2AR activity may have potential therapeutic applications in controlling ROS production by NADPH oxidase in the heart.     

Flos Puerariae Extract Prevents Myocardial Apoptosis via Attenuation Oxidative Stress in Streptozotocin-Induced Diabetic Mice

Background

Diabetic cardiomyopathy (DCM) suggests a direct cellular insult to myocardium. Apoptosis is considered as one of the hallmarks of DCM. Oxidative stress plays a key role in the pathogenesis of DCM. In this study, we explored the prevention of myocardial apoptosis by crude extract from Flos Puerariae (FPE) in experimental diabetic mice.

Methods

Experimental diabetic model was induced by intraperitoneally injection of streptozotocin (STZ, 50 mg/kg/day) for five consecutive days in C57BL/6J mice. FPE (100, 200 mg/kg) was orally administrated once a day for ten weeks. Cardiac structure changes, apoptosis, superoxide production, NADPH oxidase subunits expression (gp91^phox, p47^phox, and p67^phox), and related regulatory factors were assessed in the heart of mice.

Results

Diabetic mice were characterized by high blood glucose (≥11.1 mmol/L) and reduced body weight. In the end of the experiment, aberrant myofilament structure, as well as TUNEL positive cardiac cells coupled with increased Bax/Bcl-2 ratio and Caspase-3 expression was found in diabetic mice. Moreover, ROS formation, the ratio of NADP⁺/NADPH and NADPH oxidase subunits expression of gp91^phox and p47^phox, lipid peroxidation level was significantly increased, while antioxidant enzyme SOD and GSH-Px activity were reduced in the myocardial tissue of diabetic mice. In contrast, treatment with FPE resulted in a normalized glucose and weight profile. FPE administration also preserved myocardial structure and reduced apoptotic cardiac cell death in diabetic mice. The elevated markers of oxidative stress were significantly reversed by FPE supplementation. Further, FPE treatment markedly inhibited the increased Bax/Bcl-2 ratio and Caspase-3 expression, as well as suppressed JNK and P38 MAPK activation in the heart of diabetic mice.

Conclusions

Our data demonstrate for the first time that FPE may have therapeutic potential for STZ-induced diabetic cardiomyopathy through preventing myocardial apoptosis via attenuation oxidative stress. And this effect is probably mediated by JNK and P38 MAPK signaling pathway.     

Signaling pathways affected by mutations causing osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous connective tissue disorder characterized by bone fragility and skeletal deformity. To maintain skeletal strength and integrity, bone undergoes constant remodeling of its extracellular matrix (ECM) tightly controlled by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. There are at least 20 recognized OI-forms caused by mutations in the two collagen type I-encoding genes or genes implicated in collagen folding, posttranslational modifications or secretion of collagen, osteoblast differentiation and function, or bone mineralization. The underlying disease mechanisms of non-classical forms of OI that are not caused by collagen type I mutations are not yet completely understood, but an altered ECM structure as well as disturbed intracellular homeostasis seem to be the main defects. The ECM orchestrates local cell behavior in part by regulating bioavailability of signaling molecules through sequestration, release and activation during the constant bone remodeling process.Here, we provide an overview of signaling pathways that are associated with known OI-causing genes and discuss the impact of these genes on signal transduction. These pathways include WNT-, RANK/RANKL-, TGFβ-, MAPK- and integrin-mediated signaling as well as the unfolded protein response.     

Deficiency of biglycan causes cardiac fibroblasts to differentiate into a myofibroblast phenotype

Myocardial infarction (MI) is followed by extracellular matrix (ECM) remodeling, which is on the one hand required for the healing response and the formation of stable scar tissue. However, on the other hand, ECM remodeling can lead to fibrosis and decreased ventricular compliance. The small leucine-rich proteoglycan (SLRP), biglycan (bgn), has been shown to be critically involved in these processes. During post-infarct remodeling cardiac fibroblasts differentiate into myofibroblasts which are the main cell type mediating ECM remodeling. The aim of the present study was to characterize the role of bgn in modulating the phenotype of cardiac fibroblasts. Cardiac fibroblasts were isolated from hearts of wild-type (WT) versus bgn(-/0) mice. Phenotypic characterization of the bgn(-/0) fibroblasts revealed increased proliferation. Importantly, this phenotype of bgn(-/0) fibroblasts was abolished to the WT level by reconstitution of biglycan in the ECM. TGF-β receptor II expression and phosphorylation of SMAD2 were increased. Furthermore, indicative of a myofibroblast phenotype bgn(-/0) fibroblasts were characterized by increased α-smooth muscle actin (α-SMA) incorporated into stress fibers, increased formation of focal adhesions, and increased contraction of collagen gels. Administration of neutralizing antibodies to TGF-β reversed the pro-proliferative, myofibroblastic phenotype. In vivo post-MI α-SMA, TGF-β receptor II expression, and SMAD2 phosphorylation were markedly increased in bgn(-/0) mice. Collectively, the data suggest that bgn deficiency promotes myofibroblast differentiation and proliferation in vitro and in vivo likely due to increased responses to TGF-β and SMAD2 signaling.