IGF-II Enhances Trichostatin A-Induced TGFβ1 and p21 Expression in Hep3B Cells

Cell growth and division are controlled through the actions of cyclin-dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CKIs). Treatment of cell lines with Trichostatin A leads to induction of one of these CKIs, p21, and growth arrest. Induction of p21 can also occur through the actions of TGFβ1. Latent TGFβ1 can be activated by the M6P/IGF2R. In the present study we have examined the effect of TSA on members of the IGF axis, the CKIs p21 and p27, and also TGFβ1 in Hep3B cells. The only member of the IGF axis to be affected by treatments was IGF2. Expression of another gene from the same chromosomal location, H19, was also affected. TGFβ1 expression was greatly enhanced by TSA. In addition, both CKIs, p21 and p27, were upregulated by TSA. Effects of adding IGF-II or TGFβ1 to TSA-treated cells on p21 induction were examined. The results show that the induction of p21 by TSA can be modulated by additions of IGF-II whereas addition of TGFβ1 affects its own expression but not p21. In conclusion, the results indicate that the induction of p21 and cell growth arrest caused by Trichostatin A may involve multiple signaling pathways.     

Antagonistic Effects of TGFβ1 and BMP-6 on Skin Keratinocyte Differentiation

Several members of the transforming growth factor β (TGFβ) superfamily are expressed in the developing murine epidermis. Among these are TGFβ1, which is found in the basal layer, and bone morphogenetic protein (BMP)-6, located in the suprabasal layers. Although the role of TGFβ in cell growth has been studied extensively, little is known about the effects of these factors on keratinocyte differentiation. This study demonstrates that BMP-6 acts to positively regulate the differentiation of primary skin keratinocytes grown in culture. In contrast, TGFβ1 antagonizes keratinocyte differentiation blocking the upregulation of keratin markers by BMP-6. We show that the effects of BMP-6 on expression of keratin 1 (K1), a marker of differentiation, requires signaling through the Smad pathway. In addition, regulation of K1 levels by BMP-6 is modulated by the SEK signaling pathway. This suggests that regulation of keratinocyte differentiation by BMP-6 involves multiple signaling systems.     

Metabolic reprogramming of glycolysis and glutamine metabolism are key events in myofibroblast transition in systemic sclerosis pathogenesis

Systemic Sclerosis (SSc) is a rare fibrotic autoimmune disorder for which no curative treatments currently exist. Metabolic remodelling has recently been implicated in other autoimmune diseases; however, its potential role in SSc has received little attention. Here, we aimed to determine whether changes to glycolysis and glutaminolysis are important features of skin fibrosis. TGF‐β1, the quintessential pro‐fibrotic stimulus, was used to activate fibrotic pathways in NHDFs in vitro. Dermal fibroblasts derived from lesions of SSc patients were also used for in vitro experiments. Parameters of glycolytic function were assessed using by measuring extracellular acidification in response to glycolytic activators/inhibitors, whilst markers of fibrosis were measured by Western blotting following the use of the glycolysis inhibitors 2‐dg and 3PO and the glutaminolysis inhibitor G968. Succinate was also measured after TGF‐β1 stimulation. Itaconate was added to SSc fibroblasts and collagen examined. TGF‐β1 up‐regulates glycolysis in dermal fibroblasts, and inhibition of glycolysis attenuates its pro‐fibrotic effects. Furthermore, inhibition of glutamine metabolism also reverses TGF‐β1‐induced fibrosis, whilst glutaminase expression is up‐regulated in dermal fibroblasts derived from SSc patient skin lesions, suggesting that enhanced glutamine metabolism is another aspect of the pro‐fibrotic metabolic phenotype in skin fibrosis. TGF‐β1 was also able to enhance succinate production, with increased succinate shown to be associated with increased collagen expression. Incubation of SSc cells with itaconate, an important metabolite, reduced collagen expression. TGF‐β1 activation of glycolysis is a key feature of the fibrotic phenotype induced by TGF‐B1 in skin cells, whilst increased glutaminolysis is also evident in SSc fibroblasts.     

TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis

Gene deletion experiments have shown that the three TGFβ isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins αvβ6 and αvβ8 activate latent forms of TGFβ1 and TGFβ3. This raises the possibility that TGFβ1 and TGFβ3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFβ1 activation (Tgfb1^RGE/RGE) that were also homozygous for a null mutation in the TGFβ3 gene. Tgfb1^RGE/RGE; Tgfb3^−/− mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking αvβ8. Some Tgfb1^RGE/RGE; Tgfb3^+/− and Tgfb1^RGE/RGE; Tgfb3^+/+ mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFβ1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3^−/− mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFβ1 and TGFβ3 during development based on a shared mechanism of activation.     

The Role of SnoN in Transforming Growth Factor β1-induced Expression of Metalloprotease-Disintegrin ADAM12

Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor β1 (TGFβ1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFβ1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFβ signaling pathway, is a master regulator of ADAM12 expression in response to TGFβ1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFβ1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFβ1-induced expression of ADAM12. In a panel of TGFβ1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFβ1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFβ1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.     

Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR

The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFβ/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFβ₁ as shown by ELISA and by the inhibitory effects of a neutralizing TGFβ antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFβ type II receptor (TGFβ-RII) we furthermore demonstrate a functional involvement of both types of TGFβ receptors. However, rapamycin did not compete with TGFβ for TGFβ-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFβ/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin.     

The role of activins and follistatins in skin and hair follicle development and function

Investigations of the signalling between epithelial and mesenchymal compartments of skin during hair follicle initiation in utero and hair cycling have revealed the importance of the TGFβ superfamily in ectodermal organogenesis and morphogenesis. In particular the activins, their receptors and binding proteins such as follistatin, have been shown to be important regulators of cell proliferation, differentiation and apoptosis in hair follicle initiation, hair cycling, normal skin homeostasis and wound healing. Transgenic mice lacking various components of the activin signalling pathways display varying ectodermal pathologies including altered pelage hair follicle initiation. This review summarises the activin signal transduction pathways and the interactions between activins and other TGFβ signalling systems during hair follicle formation, hair growth cycling, skin function and wound healing.     

Phospho-specific Smad3 signaling: Impact on breast oncogenesis

Members of the TGFβ superfamily are known to exert a myriad of physiologic and pathologic growth controlling influences on mammary development and oncogenesis. In epithelial cells, TGFβ signaling inhibits cell growth through cytostatic and pro-apoptotic activities but can also induce cancer cell EMT and, thus, has a dichotomous role in breast cancer biology. Mechanisms governing this switch are the subject of active investigation. Smad3 is a critical intracellular mediator of TGFβ signaling regulated through phosphorylation by the TGFβ receptor complex at the C terminus. Smad3 is also a substrate for several other kinases that phosphorylate additional sites within the Smad protein. This discovery has expanded the understanding of the significance and complexity of TGFβ signaling through Smads. This review highlights recent advances revealing the critical role of phospho-specific Smad3 in malignancy and illustrates the potential prognostic and therapeutic impact of Smad3 phospho-isoforms in breast cancer.     

Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species

Tumour–stroma interaction is a prerequisite for tumour progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumour-associated fibroblasts to MFs (myofibroblasts) by growth factors, for example TGFβ (transforming growth factor beta(). In this study, the question was addressed of whether fibroblast-associated NAD(P)H oxidase (NADH/NADPH oxidase), known to be activated by TGFβ1, is involved in the fibroblast-to-MF switch. The up-regulation of αSMA (alpha smooth muscle actin), a biomarker for MFs, is mediated by a TGFβ1-dependent increase in the intracellular level of ROS (reactive oxygen species). This report demonstrates two novel aspects of the TGFβ1 signalling cascade, namely the generation of ROS due to a biphasic NAD(P)H oxidase activity and a ROS-dependent downstream activation of p38 leading to a transition of dermal fibroblasts to MFs that can be inhibited by the selective NAD(P)H oxidase inhibitor apocynin. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-MF switch and may be important for chemoprevention in context of a ‘stromal therapy’ which was described earlier.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

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

TGF-β3 and cancer: A review

With the development of growth factors and growth factor modulators as therapeutics for a range of disorders, it is prudent to consider whether modulating the growth factor profile in a tissue can influence tumour initiation or progression. As recombinant human TGF-β3 (avotermin) is being developed for the improvement of scarring in the skin it is important to understand the role, if any, of this cytokine in tumour progression.Elevated levels of TGF-β3 expression detected in late-stage tumours have linked this cytokine with tumourigenesis, although functional data to support a causative role are lacking. While it has proved tempting for researchers to interpret a ‘correlation’ as a ‘cause’ of disease, what has often been overlooked is the normal biological role of TGF-β3 in processes that are often subverted in tumourigenesis. Clarifying the role of this cytokine is complicated by inappropriate extrapolation of the data relating to TGF-β1 in tumourigenesis, despite marked differences in biology between the TGF-β isoforms. Indeed, published studies have indicated that TGF-β3 may actually play a protective role against tumourigenesis in a range of tissues including the skin, breast, oral and gastric mucosa. Based on currently available data it is reasonable to hypothesize that administration of acute low doses of exogenous TGF-β3 is unlikely to influence tumour initiation or progression.     

Transformation of mouse mammary epithelial cells with the Ha-ras but not with the neu oncogene results in a gene dosage-dependent increase in transforming growth factor-α production

An enhanced expression of transforming growth factor-α (TGFα) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha-ras oncogene. The amount of TGFα production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21^ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGFα secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGFα production or change in EGF receptor expression.     

MicroRNA‐411‐3p inhibits bleomycin‐induced skin fibrosis by regulating transforming growth factor‐β/Smad ubiquitin regulatory factor‐2 signalling

Skin fibrosis, which is characterized by fibroblast proliferation and increased extracellular matrix, has no effective treatment. An increasing number of studies have shown that microRNAs (miRNAs/miRs) participate in the mechanism of skin fibrosis, such as in limited cutaneous systemic sclerosis and pathological scarring. The objective of the present study was to determine the role of miR‐411‐3p in bleomycin (BLM)‐induced skin fibrosis and skin fibroblast transformation. Using Western blot analysis and real‐time quantitative polymerase chain reaction assess the expression levels of miR‐411‐3p, collagen (COLI) and transforming growth factor (TGF)‐β/Smad ubiquitin regulatory factor (Smurf)‐2/Smad signalling factors both in vitro and in vivo with or without BLM. To explore the regulatory relationship between miR‐411‐3p and Smurf2, we used the luciferase reporter assay. Furthermore, miR‐411‐3p overexpression was identified in vitro and in vivo via transfection with Lipofectamine 2000 reagent and injection. Finally, we tested the dermal layer of the skin using haematoxylin and eosin and Van Gieson''s staining. We found that miR‐411‐3p expression was decreased in bleomycin (BLM)‐induced skin fibrosis and fibroblasts. However, BLM accelerated transforming growth factor (TGF)‐β signalling and collagen production. Overexpression of miR‐411‐3p inhibited the expression of collagen, F‐actin and the TGF‐β/Smad signalling pathway factors in BLM‐induced skin fibrosis and fibroblasts. In addition, miR‐411‐3p inhibited the target Smad ubiquitin regulatory factor (Smurf)‐2. Furthermore, Smurf2 was silenced, which attenuated the expression of collagen via suppression of the TGF‐β/Smad signalling pathway. We demonstrated that miR‐411‐3p exerts antifibrotic effects by inhibiting the TGF‐β/Smad signalling pathway via targeting of Smurf2 in skin fibrosis.     

Hepsin regulates TGFβ signaling via fibronectin proteolysis

Transforming growth factor‐beta (TGFβ) is a multifunctional cytokine with a well‐established role in mammary gland development and both oncogenic and tumor‐suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent‐TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent‐TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent‐TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell‐free and cell‐based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent‐TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.