Non-canonical (non-SMAD2/3) TGF-ß signaling in fibrosis: Mechanisms and targets.

Transforming growth factor (TGF)-ß uses several intracellular signaling pathways besides canonical ALK5-Smad2/3 signaling to regulate a diverse array of cellular functions. Several of these so-called non-canonical (non-Smad2/3) pathways have been implicated in the pathogenesis of fibrosis and may therefore represent targets for therapeutic intervention. This review summarizes our current knowledge on the mechanisms of non-canonical TGF-ß signaling in fibrosis, the potential molecular targets and the use of agonists/antagonists for therapeutic intervention.

Endoglin haploinsufficiency is associated with differential regulation of extracellular matrix production during skin fibrosis and cartilage repair in mice.

Transforming growth factor (TGF)-ß is a multifunctional growth factor with potent pro-fibrotic effects. Endoglin is a TGF-ß co-receptor that strongly regulates TGF-ß signaling in a variety of cell types. Although aberrant regulation of TGF-ß signaling is known to play a key role in fibrotic diseases such as scleroderma and impaired cartilage repair, the significance of endoglin function in regulating these processes is poorly understood. Here we examined whether endoglin haploinsufficiency regulates extracellular (ECM) protein expression and fibrotic responses during bleomycin induced skin fibrosis and surgically induced osteoarthritis, using endoglin-heterozygous (Eng+/-) mice and wild-type (Eng+/+) littermates. Skin fibrosis was induced by injecting mice intradermally with bleomycin or vehicle. Osteoarthritis was induced surgically by destabilization of medial meniscus. Dermal thickness, cartilage integrity and ECM protein expression were then determined. Eng+/- mice subjected to bleomycin challenge show a marked decrease in dermal thickness (P < 0.005) and reduced collagen content and decreased collagen I, fibronectin, alpha-smooth muscle actin levels as compared to Eng+/+ mice, both under basal and bleomycin treated conditions. Eng+/- mice undergoing surgically induced osteoarthritis show no differences in the degree of cartilage degradation, as compared to Eng+/+ mice, although chondrocytes isolated from Eng+/- display markedly enhanced collagen II levels. Our findings suggest that endoglin haploinsufficiency in mice ameliorates bleomycin-induced skin fibrosis suggesting that endoglin represents a pro-fibrotic factor in the mouse skin. However, endoglin haploinsufficiency does not protect these mice from surgically indiced cartilage degradation, demonstrating differential regulation of endoglin action during skin and cartilage repair.

Overexpression of CD109 in the Epidermis Differentially Regulates ALK1 Versus ALK5 Signaling and Modulates Extracellular Matrix Synthesis in the Skin.

Transforming growth factor-ß (TGF-ß) is a multifunctional growth factor involved in many physiological processes including wound healing and inflammation. Excessive TGF-ß signaling in the skin has been implicated in fibrotic skin disorders such as keloids and scleroderma. We previously identified CD109 as a TGF-ß co-receptor and inhibitor of TGF-ß signaling and have shown that transgenic mice overexpressing CD109 in the epidermis display decreased scarring. In certain cell types, in addition to the canonical type I receptor, ALK5, which activates Smad2/3, TGF-ß can signal through another type I receptor, ALK1, which activates Smad1/5. Here we demonstrate that ALK1 is expressed and co-localizes with CD109 in mouse keratinocytes and that mice overexpressing CD109 in the epidermis display enhanced ALK1-Smad1/5 signaling but decreased ALK5-Smad2/3 signaling, TGF-ß expression, and extracellular matrix production in the skin when compared with wild-type littermates. Furthermore, treatment with conditioned media from isolated keratinocytes or epidermal explants from CD109 transgenic mouse skin leads to a decrease in extracellular matrix production in mouse skin fibroblasts. Taken together, our findings suggest that CD109 differentially regulates TGF-ß-induced ALK1-Smad1/5 versus ALK5-Smad2/3 pathways, leading to decreased extracellular matrix production in the skin and that epidermal CD109 expression regulates dermal function through a paracrine mechanism.

Transforming Growth Factor Beta Signaling in Cutaneous Wound Healing: Lessons Learned from Animal Studies.

SIGNIFICANCE: Wound healing is a complex physiological process involving a multitude of growth factors, among which transforming growth factor beta (TGF-ß) has the broadest spectrum of effects. Animal studies have provided key information on the mechanisms of TGF-ß action in wound healing and have guided the development of therapeutic strategies targeting the TGF-ß pathway to improve wound healing and scarring outcome. RECENT ADVANCES: Development of tissue-specific expression systems for overexpression or knockout of TGF-ß signaling pathway components has led to novel insight into the role of TGF-ß signaling in wound healing. This work has also identified molecules that might serve as molecular targets for the treatment of pathological skin conditions such as chronic wounds and excessive scarring (fibrosis). CRITICAL ISSUES: Many of the mouse models with genetic alterations in the TGF-ß signaling pathway develop an underlying skin abnormality, which may pose some limitations on the interpretation of wound-healing results obtained in these animals. Also, TGF-ß's pleiotropic effects on many cell types throughout all phases of wound healing present a challenge in designing specific strategies for targeting the TGF-ß signaling pathway to promote wound healing or reduce scarring. FUTURE DIRECTIONS: Further characterization of TGF-ß signaling pathway components using inducible tissue-specific overexpression or knockout technology will be needed to corroborate results obtained in mouse models that display a skin phenotype, and to better understand the role of TGF-ß signaling during distinct phases of the wound-healing process. Such studies will also provide a better understanding of how TGF-ß mediates its autocrine, paracrine, and double paracrine effects on cellular responses in vivo during wound healing.

Dynamics of Transforming Growth Factor Beta Signaling in Wound Healing and Scarring.

SIGNIFICANCE: Wound healing is an intricate biological process in which the skin, or any other tissue, repairs itself after injury. Normal wound healing relies on the appropriate levels of cytokines and growth factors to ensure that cellular responses are mediated in a coordinated manner. Among the many growth factors studied in the context of wound healing, transforming growth factor beta (TGF-ß) is thought to have the broadest spectrum of effects. RECENT ADVANCES: Many of the molecular mechanisms underlying the TGF-ß/Smad signaling pathway have been elucidated, and the role of TGF-ß in wound healing has been well characterized. Targeting the TGF-ß signaling pathway using therapeutic agents to improve wound healing and/or reduce scarring has been successful in pre-clinical studies. CRITICAL ISSUES: Although TGF-ß isoforms (ß1, ß2, ß3) signal through the same cell surface receptors, they display distinct functions during wound healing in vivo through mechanisms that have not been fully elucidated. The challenge of translating preclinical studies targeting the TGF-ß signaling pathway to a clinical setting may require more extensive preclinical research using animal models that more closely mimic wound healing and scarring in humans, and taking into account the spatial, temporal, and cell-type-specific aspects of TGF-ß isoform expression and function. FUTURE DIRECTIONS: Understanding the differences in TGF-ß isoform signaling at the molecular level and identification of novel components of the TGF-ß signaling pathway that critically regulate wound healing may lead to the discovery of potential therapeutic targets for treatment of impaired wound healing and pathological scarring.

CD109, a TGF-ß co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts.

INTRODUCTION: Scleroderma or systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of skin and internal organs. Transforming growth factor beta (TGF-ß) plays a key role in the pathogenesis of SSc fibrosis. We have previously identified CD109 as a novel TGF-ß co-receptor that inhibits TGF-ß signaling. The aim of the present study was to determine the role of CD109 in regulating extracellular matrix (ECM) production in human SSc skin fibroblasts. METHODS: CD109 expression was determined in skin tissue and cultured skin fibroblasts of SSc patients and normal healthy subjects, using immunofluorescence, western blot and RT-PCR. The effect of CD109 on ECM synthesis was determined by blocking CD109 expression using CD109-specific siRNA or addition of recombinant CD109 protein, and analyzing the expression of ECM components by western blot. RESULTS: The expression of CD109 proteinis markedly increased in SSc skin tissue in vivo and in SSc skin fibroblasts in vitro as compared to their normal counterparts. Importantly, both SSc and normal skin fibroblasts transfected with CD109-specific siRNA display increased fibronectin, collagen type I and CCN2 protein levels and enhanced Smad2/3 phosphorylation compared with control siRNA transfectants. Furthermore, addition of recombinant CD109 protein decreases TGF-ß1-induced fibronectin, collagen type I and CCN2 levels in SSc and normal fibroblasts. CONCLUSION: The upregulation of CD109 protein in SSc may represent an adaptation or consequence of aberrant TGF-ß signaling in SSc. Our finding that CD109 is able to decrease excessive ECM production in SSc fibroblasts suggest that this molecule has potential therapeutic value for the treatment of SSc.

Characterization of Wnt2 overexpression in a rat granulosa cell line (DC3): effects on CTNNB1 activation.

WNTs comprise a family of secreted glycoproteins that are essential for normal embryonic development of the female reproductive system. The functional role that WNTs play in the postnatal ovary is poorly defined. We have shown previously that Wnt2 and Fzd4 mRNAs are expressed in granulosa cells of the postnatal rat ovary. Here we examine the effects of Wnt2 overexpression in a rat granulosa cell line (DC3) that displays characteristics of granulosa cells at an early stage of follicular development. We show that DC3 cells express a 7.7-kb Fzd4 mRNA transcript similar in size to that detected in the rat and human ovary. Our results demonstrate that Wnt2 overexpression in DC3 promotes cytosolic and nuclear accumulation of beta-catenin (CTNNB1), but does not stimulate CTNNB1/TCF-dependent (pGL3-OT) transcriptional activity. We show that chibby (CBY1), a nuclear CTNNB1-associated antagonist of the WNT pathway, is expressed in DC3 cells and associates with CTNNB1 in the presence and absence of Wnt2 overexpression, suggesting that Cby1 contributes to suppression of CTNNB1/TCF-dependent transcription in these cells. Our results show that Wnt2 overexpression in DC3 cells increases follistatin (Fst) mRNA expression and promotes resistance to activin-induced cell deletion. Taken together, our results suggest that WNT2 opposes activin activity in granulosa cells by up-regulating expression of the activin antagonist Fst in a CTNNB1/TCF-independent manner, and that rat granulosa cells express factors, including Cby1, that suppress CTNNB1/TCF-dependent signal transduction in the presence of a WNT signal.

TGF-b signaling in cartilage homeostasis and osteoarthritis.

Healthy cartilage is maintained by a delicate balance between the anabolic and catabolic activities of articular chondrocytes. This involves actions of numerous cytokines and growth factors that regulate the synthesis and degradation of extracellular matrix components which maintain the functional integrity of the joint. An imbalance between the activities of these anabolic and catabolic factors leads to cartilage degradation resulting in osteoarthritis (OA), a chronic degenerative joint disorder characterized by destruction of articular cartilage, alterations of subchondral bone and synovial fibrosis. Among the cytokines and growth factors that have been studied in the context of cartilage homeostasis and OA, transforming growth factor-beta TGF-beta has emerged as an important molecule that plays a critical role in the development, growth, maintenance and repair of articular cartilage. Deregulation of its signaling and responses has been shown to be involved in OA. Several components of the TGF-beta pathway, including extracellular, cell surface and intracellular molecules, display altered expression or action in OA. In this review, we discuss the regulatory mechanisms of TGF-beta signaling and link these mechanisms to cartilage function, highlighting the important role of TGF-beta in maintaining cartilage function and integrity. We also summarize the alterations in the molecular events of TGF-beta signaling and responses that may contribute to OA progression and discuss the potential of targeting the TGF-beta signaling pathway for the development of novel therapies for OA.

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)-ß represents an important pro-fibrogenic factor and aberrant TGF-ß action has been implicated in many disease processes of the liver. Endoglin is a TGF-ß co-receptor expressed mainly in endothelial cells that has been shown to differentially regulates TGF-ß 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-ß 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-ß-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-ß 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.

The TGF-ß co-receptor, CD109, promotes internalization and degradation of TGF-ß receptors.

Transforming growth factor-ß (TGF-ß) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-ß receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-ß action. We previously identified CD109, a GPI-anchored protein, as a TGF-ß co-receptor and a negative regulator of TGF-ß signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-ß to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-ß receptors into the caveolar compartment in the presence of ligand and facilitates TGF-ß-receptor degradation. Thus, CD109 regulates TGF-ß receptor endocytosis and degradation to inhibit TGF-ß signaling. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

ALK1 opposes ALK5/Smad3 signaling and expression of extracellular matrix components in human chondrocytes.

INTRODUCTION: TGF-beta is a multifunctional regulator of chondrocyte proliferation, differentiation, and extracellular matrix production. Dysregulation of TGF-beta action has been implicated in cartilage diseases such as osteoarthritis. TGF-beta signaling is transduced through a pair of transmembrane serine/threonine kinases, known as the type I (ALK5) and type II receptors. However, recent studies on endothelial cells have identified ALK1 as a second type I TGF-beta receptor and have shown that ALK1 and ALK5 have opposing functions in these cells. Here we examined ALK1 expression and its regulation of TGF-beta signaling and responses in human chondrocytes. MATERIALS AND METHODS: ALK1 expression in human chondrocytes was examined by RT-PCR and Western blot. The ability of ALK1 to form complexes with other TGF-beta receptors was determined by affinity labeling/immunoprecipitation and by immunoprecipitation followed by Western blot. The effect of ALK1 on TGF-beta1-induced signaling and responses was determined by varying ALK1 expression levels and measuring transcriptional activity using promoter/luciferase assays, Smad1/5 and Smad3 phosphorylation, and expression of type II collagen, PAI-1, and fibronectin. RESULTS: Our results indicate that ALK1 is expressed in human chondrocytes and that it is a component of the TGF-beta receptor system, associating with ALK5, type II TGF-beta receptor, endoglin, and betaglycan. Furthermore, we show that both ALK1 and ALK5 are needed for TGF-beta-induced phosphorylation of intracellular mediators Smad1/5, whereas only ALK5 is essential for TGF-beta1-induced phosphorylation of Smad3. In addition, our results show that ALK1 inhibits, whereas ALK5 potentiates, TGF-beta-induced Smad3-driven transcriptional activity and the expression of PAI-1, fibronectin, and type II collagen in chondrocytes. CONCLUSIONS: Our results suggest that ALK1 and ALK5 display opposing functions in human chondrocytes, implicating an essential role for ALK1 in the regulation of TGF-beta signaling and function in these cells.

Identification of CD109 as part of the TGF-beta receptor system in human keratinocytes.

We have previously reported that keratinocytes defective in glycosylphosphatidylinositol (GPI)-anchor biosynthesis display enhanced TGF-beta responses. These studies implicated the involvement of a 150 kDa GPI-anchored TGF-beta1 binding protein, r150, in modulating TGF-beta signaling. Here, we sought to determine the molecular identity of r150 by affinity purification and microsequencing. Our results identify r150 as CD109, a novel member of the alpha2-macroglobulin (alpha2M)/complement superfamily, whose function has remained obscure. In addition, we have identified a novel CD109 isoform that occurs in the human placenta but not keratinocytes. Biochemical studies show that r150 contains an internal thioester bond, a defining feature of the alpha2M/complement family. Loss and gain of function studies demonstrate that CD109 is a component of the TGF-beta receptor system, and a negative modulator of TGF-beta responses in keratinocytes, as implicated for r150. Our data suggest that CD109 can inhibit TGF-beta signaling independently of ligand sequestration and may exert its effect on TGF-beta signaling by direct modulation of receptor activity. Together, our results linking CD109 function to regulation of TGF-beta signaling suggest that CD109 plays a unique role in the regulation of isoform-specific TGF-beta signaling in keratinocytes.

Glycosylphosphatidylinositol-anchored proteins regulate transforming growth factor-beta signaling in human keratinocytes.

Glycosylphosphatidylinositol (GPI)-anchored proteins have been demonstrated to bind transforming growth factor-beta (TGF-beta) in certain cell lines. However, the identity of these GPI-anchored proteins and the role they may play in TGF-beta signaling remain unknown. We have previously reported the presence of GPI-anchored TGF-beta-binding proteins on human skin fibroblasts and keratinocytes (Tam, B. Y. Y., and Philip, A. (1998) J. Cell. Physiol. 176, 553-564; Tam, B. Y. Y., Germain, L., and Philip, A. (1998) J. Cell. Biochem. 70, 573-586). On human keratinocytes, we identified a 150-kDa GPI-anchored TGF-beta1-binding protein (r150) and demonstrated that it can form a heteromeric complex with the type I and II TGF-beta signaling receptors. To explore whether GPI-anchored proteins modulate TGF-beta signaling in keratinocytes, we created keratinocytes defective in GPI anchor biosynthesis (GPI mutant cells) by chemical mutagenesis of HaCaT cells. Mutant clones were selected by fluorescence-activated cell sorting analysis based on the loss of a CD59 marker. In comparison with parental HaCaT cells, GPI mutant cells demonstrated a significant loss of r150 expression. In contrast, the levels of the type I and II TGF-beta receptors and their ligand affinities, cell morphology, and doubling time remained unchanged. Importantly, GPI mutant cells displayed enhanced gene transcriptional activity and Smad2 and Smad3 activation in response to TGF-beta1 treatment in a dose-dependent manner. Taken together, our results indicate that GPI-anchored protein(s) inhibit TGF-beta signaling and implicate r150 as the GPI-anchored protein responsible for this inhibition in human keratinocytes.

Expression of multiple connexins in the rat epididymis indicates a complex regulation of gap junctional communication.

In the epididymis, Cx43 forms gap junctions between principal and basal cells but not between adjacent principal cells. Cx30.3, 31.1, and 32 were identified in adult rat epididymis by RT-PCR, whereas Cx26 was present in young rats. Postnatal development studies indicate that Cx26 mRNA was detectable only in the caput-corpus region of the epididymis and that levels increased by fivefold during the first 4 wk postnatally, when epithelial cells differentiate, and decrease to nondetectable levels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout the epididymis in young rats and began to increase in the second and third weeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32 were localized to the lateral plasma membranes between adjacent epithelial cells of the epididymis. Colocalization studies indicate that Cx26 and Cx32 exist either independently of one another or can colocalize along the lateral plasma membrane of epithelial cells in young rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.

Thyroid toxicity due to subchronic exposure to a complex mixture of 16 organochlorines, lead, and cadmium.

The human population in the industrialized world is ubiquitously exposed to complex mixtures of persistent pollutants that contaminate food, water, and air. A large number of these contaminants have been shown to cause significant toxicity to the hypothalamic-pituitary-thyroid (HPT) axis in laboratory animal studies, through a variety of mechanisms, although these effects occur at levels of exposure greatly in excess of common human exposure. While many of the mechanisms of thyroid toxicity of these substances are potentially complementary, little is known of the degree of interaction of common persistent contaminants on responses of the HPT axis. To investigate the potential effects of a complex, environmentally relevant mixture on the HPT axis, sexually mature male rats were administered a mixture of 16 common organochlorines (dichlorodiphenoxytrichloroethane [DDT], p,p'-dichlorodiphenoxydichloroethylene [p,p'-DDE], hexachlorobenzene [HCB], tetrachlorodibenzo-p-dioxin [TCDD], polychlorinated biphenyls [PCBs], methoxychlor, endosulfan, heptachlor, hexachlorocyclohexane, dieldrin, aldrin, mirex, and several chlorinated benzenes, and metal contaminants [lead, cadmium]). The doses of the mixture that were administered were related to minimum risk levels or tolerable daily intakes of these substances, as derived by risk assessment with the 1x, 10x, 100x, and 1000x groups receiving mixture components at doses equivalent to 1x, 10x, 100x, or 1000x the minimum risk level (or tolerable daily intake, reference dose), respectively. After 70 daily treatments by gavage, endpoints related to circulating thyroid hormone (serum thyroxine [T(4)], triiodothyronine [T(3)], thyroid stimulating hormone [TSH], and serum T(3) uptake [T(3)-up]), thyroid gland histomorphology (thyroid follicle cross sectional area, epithelial height, follicle roundness or aspect ratio, colloid/epithelial ratio) and hepatic metabolism of thyroid hormone (UDP-glucuronyl transferase [UGT] and outer-ring deiodinase [ORD]) were assessed. All examined endpoints were significantly altered by the mixture albeit with great variability between endpoints in the sensitivity. While most endpoints examined did not show significant changes at mixture doses below 1000x, 2 endpoints, TSH and hepatic outer ring deiodinase activity, were significantly increased and decreased, respectively, by 1x dose and showed dose-related increases in severity with increasing dose. Median thyroid follicle cross sectional area was also increased by the lowest dose of the mixture but decreased with subsequent increases in dose until, at the highest dose, this parameter was significantly reduced relative to control. The relative sensitivity of endpoints of thyroid function in detecting toxicity of the mixture was TSH = ORD = median follicle area >> T(3) > all other endpoints. These results demonstrate that low doses of ubiquitous environmental contaminants can alter HPT physiology in sexually mature males.