HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation

Transforming growth factor-β (TGF-β) signaling plays a key role in excessive fibrosis. As a class IIa family histone deacetylase (HDAC), HDAC5 shows a close relationship with TGF-β signaling and fibrosis. However, the effect and regulatory mechanism of HDAC5 in hypertrophic scar (HS) formation remain elusive. We show that HDAC5 was overexpressed in HS tissues and depletion of HDAC5 attenuated HS formation in vivo and inhibited fibroblast activation in vitro. HDAC5 knockdown (KD) significantly downregulated TGF-β1 induced Smad2/3 phosphorylation and increased Smad7 expression. Meanwhile, Smad7 KD rescued the Smad2/3 phosphorylation downregulation and scar hyperplasia inhibition mediated by HDAC5 deficiency. Luciferase reporter assays and ChIP-qPCR assays revealed that HDAC5 interacts with myocyte enhancer factor 2A (MEF2A) suppressing MEF2A binding to the Smad7 promoter region, which results in Smad7 promoter activity repression. HDAC4/5 inhibitor, LMK235, significantly alleviated hypertrophic scar formation. Our study provides clues for the development of HDAC5 targeting strategies for the therapy or prophylaxis of fibrotic diseases.     

Endoglin in liver fibrosis

Liver fibrosis occurs in most types of chronic liver diseases and is characterized by excessive accumulation of extracellular matrix proteins, leading to disruption of tissue function and eventually organ failure. Transforming growth factor (TGF)-β represents an important pro-fibrogenic factor and aberrant TGF-β action has been implicated in many disease processes of the liver. Endoglin is a TGF-β co-receptor expressed mainly in endothelial cells that has been shown to differentially regulates TGF-β signal transduction by inhibiting ALK5-Smad2/3 signalling and augmenting ALK1-Smad1/5 signalling. Recent reports demonstrating upregulation of endoglin expression in pro-fibrogenic cell types such as scleroderma fibroblasts and hepatic stellate cells have led to studies exploring the potential involvement of this TGF-β co-receptor in organ fibrosis. A recent article by Meurer and colleagues now shows that endoglin expression is increased in transdifferentiating hepatic stellate cells in vitro and in two different models (carbon tetrachloride intoxication and bile duct ligation) of liver fibrosis in vivo. Moreover, they show that endoglin overexpression in hepatic stellate cells is associated with enhanced TGF-β-driven Smad1/5 phosphorylation and α-smooth muscle actin production without altering Smad2/3 signaling. These findings suggest that endoglin may play an important role in hepatic fibrosis by altering the balance of TGF-β signaling via the ALK1-Smad1/5 and ALK-Smad2/3 pathways and raise the possibility that targeting endoglin expression in transdifferentiating hepatic stellate cells may represent a novel therapeutic strategy for the treatment of liver fibrosis.     

TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage

Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-beta/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3(ex8/ex8)) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-beta1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3(ex8/ex8) mice. These data suggest that TGF-beta/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.     

The application of MRI for depiction of subtle blood brain barrier disruption in stroke

The development of imaging methodologies for detecting blood-brain-barrier (BBB) disruption may help predict stroke patient's propensity to develop hemorrhagic complications following reperfusion. We have developed a delayed contrast extravasation MRI-based methodology enabling real-time depiction of subtle BBB abnormalities in humans with high sensitivity to BBB disruption and high spatial resolution. The increased sensitivity to subtle BBB disruption is obtained by acquiring T1-weighted MRI at relatively long delays (~15 minutes) after contrast injection and subtracting from them images acquired immediately after contrast administration. In addition, the relatively long delays allow for acquisition of high resolution images resulting in high resolution BBB disruption maps. The sensitivity is further increased by image preprocessing with corrections for intensity variations and with whole body (rigid+elastic) registration. Since only two separate time points are required, the time between the two acquisitions can be used for acquiring routine clinical data, keeping the total imaging time to a minimum. A proof of concept study was performed in 34 patients with ischemic stroke and 2 patients with brain metastases undergoing high resolution T1-weighted MRI acquired at 3 time points after contrast injection. The MR images were pre-processed and subtracted to produce BBB disruption maps. BBB maps of patients with brain metastases and ischemic stroke presented different patterns of BBB opening. The significant advantage of the long extravasation time was demonstrated by a dynamic-contrast-enhancement study performed continuously for 18 min. The high sensitivity of our methodology enabled depiction of clear BBB disruption in 27% of the stroke patients who did not have abnormalities on conventional contrast-enhanced MRI. In 36% of the patients, who had abnormalities detectable by conventional MRI, the BBB disruption volumes were significantly larger in the maps than in conventional MRI. These results demonstrate the advantages of delayed contrast extravasation in increasing the sensitivity to subtle BBB disruption in ischemic stroke patients. The calculated disruption maps provide clear depiction of significant volumes of BBB disruption unattainable by conventional contrast-enhanced MRI.     

Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1

Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix.     

Characterization of type III TGF-β receptor expression in invasive breast carcinomas: a potential new marker and target for triple negative breast cancer

Invasive breast carcinomas are heterogeneous and exhibit distinct molecular features and biological behavior. Understanding the underlying molecular events that promote breast cancer progression is necessary to improve treatment and prognostication. TGF-β receptor III (TBR3) is a member of the TGF-β signaling pathway, with functions in cell proliferation and migration in malignancies, including breast cancer. Recent studies propose that TBR3 may function as a tumor suppressor and that its loss may correlate with disease progression. However, there are limited data on the expression of TBR3 in breast cancer in relationship to tumor type, hormonal receptor status and HER-2/neu, and patient outcome. In this study, we investigated the expression of TBR3 in a cohort of 205 primary invasive breast carcinomas in tissue microarrays (TMAs), with comprehensive clinical, pathological and follow- up information. Sections were stained for TBR3 and evaluated for intensity of reactivity based on a 4-tiered scoring system (1 to 4; TBR3 low = scores 1–2; TBR3 high = scores 3–4). Of the 205 invasive carcinomas, 123 were luminal type (95 type A, 28 type B), 8 were HER-2 type, and 62 were triple negative (TN). TBR3 was high in 112 (55 %) and low in 93 (45 %) cases. Low TBR3 was associated with higher histological grade and worse disease free and overall survival, all features of biologically aggressive breast carcinomas. TBR3 was significantly associated with the subtype of breast cancer, as low TBR3 was detected in 95 % of TN compared to 22 % of luminal tumors (p < 0.0001). We discovered a significant association between low TBR3 protein expression, TN breast cancer phenotype, and disease progression. These data suggest that TBR3 loss might be linked to the development of TN breast cancers and pave the way to investigating whether restoring TBR3 function may be a therapeutic strategy against TN breast carcinomas.     

Extraction and purification of decorin from corneal stroma retain structure and biological activity

We developed a method to purify decorin core protein from tissue with the goal of preserving its native structure and biological function. Currently, most procedures rely on the use of denaturing reagents potentially altering the biological activity. Decorin was purified from corneal stromas without the use of detergents or chaotropic reagents. Proteoglycans isolated using anion exchange chromatography on Q-Sepharose were treated with chondroitinase ABC. Decorin was isolated by a second Q-Sepharose chromatography with affinity chromatographies on heparin-Sepharose and concanavalin A-Sepharose. SDS-PAGE revealed a 98.4% pure 44kDa protein identified as decorin with a yield of 35mg per 100 bovine corneas. Identification was confirmed by NanoESI and MALDI qTOF. The novel inclusion of 20% propylene glycol in extraction and column buffers resulted in recoveries of proteoglycans comparable with those observed with detergents and urea. Purified decorin did alter the rate of fibrillogenesis of type I collagen and inhibited the lateral fusion of collagen fibrils. It also bound to [125I]TGF-beta1 with an apparent K(d) of 40nM. Circular dichroism spectroscopy of decorin displayed the spectra of alpha-helices and beta-pleated sheets consistent with those obtained from recombinant decorin. Urea-induced unfolding was cooperative and reversible while thermal denaturation caused irreversible unfolding. Native decorin can be purified from tissue in quantity and quality for biophysical, biochemical, and biological assays.     

Smad3-mediated myocardin silencing: a novel mechanism governing the initiation of smooth muscle differentiation

Both TGF-β and myocardin (MYOCD) are important for smooth muscle cell (SMC) differentiation, but their precise role in regulating the initiation of SMC development is less clear. In TGF-β-induced SMC differentiation of pluripotent C3H10T1/2 progenitors, we found that TGF-β did not significantly induce Myocd mRNA expression until 18 h of stimulation. On the other hand, early SMC markers such as SM α-actin, SM22α, and SM calponin were detectable beginning 2 or 4 h after TGF-β treatment. These results suggest that Myocd expression is blocked during the initiation of TGF-β-induced SMC differentiation. Consistent with its endogenous expression, Myocd promoter activity was not elevated until 18 h following TGF-β stimulation. Surprisingly, Smad signaling was inhibitory to Myocd expression because blockade of Smad signaling enhanced Myocd promoter activity. Overexpression of Smad3, but not Smad2, inhibited Myocd promoter activity. Conversely, shRNA knockdown of Smad3 allowed TGF-β to activate the Myocd promoter in the initial phase of induction. Myocd was activated by PI3 kinase signaling and its downstream target Nkx2.5. Interestingly, Smad3 did not affect PI3 kinase activity. However, Smad3 physically interacted with Nkx2.5. This interaction blocked Nkx2.5 binding to the Myocd promoter in the early stage of TGF-β induction, leading to inhibition of Myocd mRNA expression. Moreover, Smad3 inhibited Nkx2.5-activated Myocd promoter activity in a dose-dependent manner. Taken together, our results reveal a novel mechanism for Smad3-mediated inhibition of Myocd in the initiation phase of SMC differentiation.     

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.     

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.     

Tsukushi is involved in the wound healing by regulating the expression of cytokines and growth factors

During the wound-healing process, macrophages, fibroblasts, and myofibroblasts play a leading role in shifting from the inflammation phase to the proliferation phase, although little is known about the cell differentiation and molecular control mechanisms underlying these processes. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions as a key extracellular coordinator of multiple signalling networks. In this study, we investigated the contribution of TSK to wound healing. Analysis of wound tissue in heterozygous TSK-lacZ knock-in mice revealed a pattern of sequential TSK expression from macrophages to myofibroblasts. Quantitative PCR and in vitro cell induction experiments showed that TSK controls macrophage function and myofibroblast differentiation by inhibiting TGF-β1 secreted from macrophages. Our results suggest TSK facilitates wound healing by maintaining inflammatory cell quiescence.     

Bone abnormalities in latent TGF-[beta] binding protein (Ltbp)-3-null mice indicate a role for Ltbp-3 in modulating TGF-[beta] bioavailability.

The TGF-betas are multifunctional proteins whose activities are believed to be controlled by interaction with the latent TGF-beta binding proteins (LTBPs). In spite of substantial effort, the precise in vivo significance of this interaction remains unknown. To examine the role of the Ltbp-3, we made an Ltbp-3-null mutation in the mouse by gene targeting. Homozygous mutant animals develop cranio-facial malformations by day 10. At 2 mo, there is a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. Histological examination of the skulls from null animals revealed ossification of the synchondroses within 2 wk of birth, in contrast to the wild-type synchondroses, which never ossify. Between 6 and 9 mo of age, mutant animals also develop osteosclerosis and osteoarthritis. The pathological changes of the Ltbp-3-null mice are consistent with perturbed TGF-beta signaling in the skull and long bones. These observations give support to the notion that LTBP-3 is important for the control of TGF-beta action. Moreover, the results provide the first in vivo indication for a role of LTBP in modulating TGF-beta bioavailability.     

Complexities of TGF-β targeted cancer therapy

Many advanced tumors produce excessive amounts of Transforming Growth Factor-β (TGF-β) which, in normal epithelial cells, is a potent growth inhibitor. However, in oncogenically activated cells, the homeostatic action of TGF-β is often diverted along alternative pathways. Hence, TGF-β signaling elicits protective or tumor suppressive effects during the early growth-sensitive stages of tumorigenesis. However, later in tumor development when carcinoma cells become refractory to TGF-β-mediated growth inhibition, the tumor cell responds by stimulating pathways with tumor progressing effects. At late stages of malignancy, tumor progression is driven by TGF-β overload. The tumor microenvironment is a target of TGF-β action that stimulates tumor progression via pro-tumorigenic effects on vascular, immune, and fibroblastic cells. Bone is one of the richest sources of TGF-β in the body and a common site for dissemination of breast cancer metastases. Osteoclastic degradation of bone matrix, which accompanies establishment and growth of metastases, triggers further release of bone-derived TGF-β. This leads to a vicious positive feedback of tumor progression, driven by ever increasing levels of TGF-β released from both the tumor and bone matrix. It is for this reason, that pharmaceutical companies have developed therapeutic agents that block TGF-β signaling. Nonetheless, the choice of drug design and dosing strategy can affect the efficacy of TGF-β therapeutics. This review will describe pre-clinical and clinical data of four major classes of TGF-β inhibitor, namely i) ligand traps, ii) antisense oligonucleotides, iii) receptor kinase inhibitors and iv) peptide aptamers. Long term dosing strategies with TGF-β inhibitors may be ill-advised, since this class of drug has potentially highly pleiotropic activity, and development of drug resistance might potentiate tumor progression. Current paradigms for the use of TGF-β inhibitors in oncology have therefore moved towards the use of combinatorial therapies and short term dosing, with considerable promise for the clinic.     

Pancreatic cancer cells respond to type I collagen by inducing snail expression to promote membrane type 1 matrix metalloproteinase-dependent collagen invasion

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.     

Regulation of pro-inflammatory and pro-fibrotic factors by CCN2/CTGF in H9c2 cardiomyocytes

Connective tissue growth factor (CTGF), also known as CCN2, is implicated in fibrosis through both extracellular matrix (ECM) induction and inhibition of ECM degradation. The role of CTGF in inflammation in cardiomyocytes is unknown. In some mesenchymal cell systems, CTGF mediates effects through TGF-β or tyrosine kinase cell surface receptor, TrkA, signalling. In this study, cellular mechanisms by which CTGF regulates pathways involved in fibrosis and inflammation were explored. Murine H9c2 cardiomyocytes were treated with recombinant human (rh)CTGF and ECM formation gene expression: fibronectin, collagen type -I and -III and ECM degradation genes: TIMP-1, TIMP-2 and PAI-1 were found to be induced. CTGF treatment also increased pro-inflammatory cytokines TNF-α, IL-6, MCP-1 and IL-8. CTGF upregulated TGF-β1 mRNA and rapidly induced phosphorylation of TrkA. The CTGF-induced pro-fibrotic and pro-inflammatory effects were blocked by anti-TGF-β neutralizing antibody and Alk 5 inhibitor (SB431542). A specific blocker of TrkA activation, k252a, also abrogated CTGF-induced effects on fibrosis and gene expresison of MCP-1 and IL-8, but not TNF-α or IL-6. Collectively, this data implicates CTGF in effects on pro-fibrotic genes and pro-inflammatory genes via TGF-β pathway signalling and partly through TrkA.     

Dual roles of immune cells and their factors in cancer development and progression

Traditional wisdom holds that intact immune responses, such as immune surveillance or immunoediting, are required for preventing and inhibiting tumor development; but recent evidence has also indicated that unresolved immune responses, such as chronic inflammation, can promote the growth and progression of cancer. Within the immune system, cytotoxic CD8(+) and CD4(+) Th1 T cells, along with their characteristically produced cytokine IFN-γ, function as the major anti-tumor immune effector cells, whereas tumor associated macrophages (TAM) or myeloid-derived suppressive cells (MDSC) and their derived cytokines IL-6, TNF, IL-1β and IL-23 are generally recognized as dominant tumor-promoting forces. However, the roles played by Th17 cells, CD4(+) CD25(+) Foxp3(+) regulatory T lymphocytes and immunoregulatory cytokines such as TGF-β in tumor development and survival remain elusive. These immune cells and the cellular factors produced from them, including both immunosuppressive and inflammatory cytokines, play dual roles in promoting or discouraging cancer development, and their ultimate role in cancer progression may rely heavily on the tumor microenvironment and the events leading to initial propagation of carcinogenesis.