Kenneth W. Finnson

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

Introduction: TGF‐β is a multifunctional regulator of chondrocyte proliferation, differentiation, and extracellular matrix production. Dysregulation of TGF‐β action has been implicated in cartilage diseases such as osteoarthritis. TGF‐β 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‐β receptor and have shown that ALK1 and ALK5 have opposing functions in these cells. Here we examined ALK1 expression and its regulation of TGF‐β signaling and responses in human chondrocytes.

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

We have previously reported that keratinocytes defective in glycosylphosphatidylinositol (GPI)‐anchor biosynthesis display enhanced TGF‐β responses. These studies implicated the involvement of a 150 kDa GPI‐anchored TGF‐β1 binding protein, r150, in modulating TGF‐β 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 α2‐macroglob‐ulin (α2M)/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 α2M/complement family. Loss and gain of function studies demonstrate that CD109 is a component of the TGF‐β receptor system, and a negative modulator of TGF‐β responses in keratinocytes, as implicated for r150. Our data suggest that CD109 can inhibit TGF‐β signaling independently of ligand sequestration and may exert its effect on TGF‐β signaling by direct modulation of receptor activity. Together, our results linking CD109 function to regulation of TGF‐β signaling suggest that CD109 plays a unique role in the regulation of isoform‐specific TGF‐β signaling in keratinocytes.—Finnson, K. W., Tam, B. Y. Y., Liu, K., Marcoux, A., Lepage, P., Roy, S., Bizet, A. A., Philip, A. Identification of CD109 as a TGF‐β1 accessory receptor in human keratinocytes. FASEB J. 20, E780–E795 (2006)

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls.

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

Transforming growth factor-β (TGF-β) 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-β 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-β action. We previously identified CD109, a GPI-anchored protein, as a TGF-β co-receptor and a negative regulator of TGF-β signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-β to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand and facilitates TGF-β-receptor degradation. Thus, CD109 regulates TGF-β receptor endocytosis and degradation to inhibit TGF-β signaling. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes

OBJECTIVE Transforming growth factor-β (TGF-β) plays a critical role in cartilage homeostasis and deregulation of its signalling is implicated in osteoarthritis (OA). TGF-β isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-β receptors. Endoglin is a TGF-β co-receptor that binds TGF-β with high affinity in the presence of the type II TGF-β receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-β signalling receptors. However, the functional significance of endoglin expression in chondrocytes is unknown. Our objective was to determine whether endoglin regulates TGF-β/Smad signalling and extracellular matrix (ECM) production in human chondrocytes and whether its expression varies with chondrocyte differentiation state. METHOD Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-β-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples. RESULTS Endoglin enhances TGF-β1-induced Smad1/5 phosphorylation and inhibits TGF-β1-induced Smad2 phosphorylation, Smad3-driven transcriptional activity and ECM production in human chondrocytes. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-β1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage. CONCLUSION Together, our results suggest that endoglin regulates the balance between TGF-β/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes and that endoglin may represent a marker for chondrocyte phenotype.

TGF-beta 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.

BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

BackgroundAxolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas) in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration.ResultsThe expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration) but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor) in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division.ConclusionThe expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has described in other tetrapods. Overexpression of BMP-2 did not cause the formation of extra digits, which is consistent with the hypothesis that it is not the secondary signal of sonic hedgehog. However, it did cause the formation of hypomorphic limbs as a result of increased cellular condensation and apoptosis. Taken together, these results suggest that BMP-2 does not have a direct role in patterning regenerating limbs but may be important to trigger condensation prior to ossification and to mediate apoptosis.

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

IntroductionScleroderma or systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of skin and internal organs. Transforming growth factor beta (TGF-β) plays a key role in the pathogenesis of SSc fibrosis. We have previously identified CD109 as a novel TGF-β co-receptor that inhibits TGF-β 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.MethodsCD109 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.ResultsThe 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-β1-induced fibronectin, collagen type I and CCN2 levels in SSc and normal fibroblasts.ConclusionThe upregulation of CD109 protein in SSc may represent an adaptation or consequence of aberrant TGF-β 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.

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.

Characterization of Wnt2 Overexpression in a Rat Granulosa Cell Line (DC3): Effects on CTNNB1 Activation

ABSTRACT 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.