Wnt-mediated regulation of chondrocyte maturation: modulation by TGF-beta

Wnt proteins are expressed during limb morphogenesis, yet their role and mechanism of action remains unclear during long bone growth. Wnt expression, effects and modulation of signaling events by BMP and transforming growth factor-beta (TGF-beta) were evaluated in chick embryonic chondrocytes. Chondrocyte cell cultures underwent spontaneous maturation with increased expression of colX and this was associated with an increase in the expression of multiple Wnts, including Wnt 4, 5a, 8c, and 9a. Both parathyroid hormone related peptide (PTHrP) and TGF-beta inhibited colX, but had disparate effects on Wnt expression. While TGF-beta strongly inhibited all Wnts, PTHrP did not inhibit either Wnt8c or Wnt9a and had lesser effects on the expression of the other Wnts. BMP-2 induced colX expression, and also markedly increased Wnt8c expression. Overexpression of beta-catenin and/or T cell factor (TCF)-4 also induced the type X collagen promoter. Overexpression of Wnt8c induced maturation, as did overexpression of beta-catenin. The Wnt8c/beta-catenin maturational effects were enhanced by BMP-2 and inhibited by TGF-beta. TGF-beta also inhibited activation of the Topflash reporter by beta-catenin, suggesting a direct inhibitory effect since the Topflash reporter contains only beta-catenin binding sequences. In turn beta-catenin inhibited activation of the p3TP-Luc reporter by TGF-beta, although the effect was partial. Thus, Wnt/beta-catenin signaling is a critical regulator of the rate of chondrocyte differentiation. Moreover, this pathway is modulated by members of the TGF-beta family and demonstrates the highly integrated nature of signals controlling endochondral ossification.     

Transforming growth factor-beta isoforms differently stimulate proalpha2 (I) collagen gene expression during wound healing process in transgenic mice

The role of many growth factors and cytokines in the process of wound healing has been intensively investigated in recent two decades. Among them, transforming growth factor-betas (TGF-betas) are well known to have a potent stimulatory effect on collagen synthesis as shown in various in vivo experimental systems. In the present study, we examined the effects of various growth factors on the promoter activity of the proalpha2 (I) collagen gene (COL1A2) during the wound healing process. For this purpose, we utilized transgenic mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase or a bacterial beta-galactosidase gene. These mice exhibited normal phenotypic expression and the wound healing process was not impaired. Full thickness wounds were made by punch biopsy. We examined the effects of TGF-beta1, -beta2, -beta3, basic fibroblast growth factor, platelet-derived growth factor, and connective tissue growth factor by applying them locally to the open wound every 2 days. Among the growth factors examined, all of the three isoforms of TGF- exhibited a more potent stimulatory effect on COL1A2 promoter activity than did other factors. In addition, while TGF-beta1 and -beta2 significantly increased the number of fibroblasts which were positive for X-Gal staining, TGF-beta3 treatment did not change the number of beta-galactosidase expressing cells. Accumulation of collagen fibers was observed to the same extent in the mice treated with TGF-beta1 and those with TGF-beta3. These findings suggest that TGF-beta1 and -beta3 have similar but not identical regulatory mechanisms of COL1A2 expression, and that their pathophysiological roles in wound healing might be different from each other.     

Abnormal lens morphogenesis and ectopic lens formation in the absence of beta-catenin function

beta-Catenin plays a key role in cadherin-mediated cell adhesion as well as in canonical Wnt signaling. To study the role of beta-catenin during eye development, we used conditional Cre/loxP system in mouse to inactivate beta-catenin in developing lens and retina. Inactivation of beta-catenin does not suppress lens fate, but instead results in abnormal morphogenesis of the lens. Using BAT-gal reporter mice, we show that beta-catenin-mediated Wnt signaling is notably absent from lens and neuroretina throughout eye development. The observed defect is therefore likely due to the cytoskeletal role of beta-catenin, and is accompanied by impaired epithelial cell adhesion. In contrast, inactivation of beta-catenin in the nasal ectoderm, an area with active Wnt signaling, results in formation of crystallin-positive ectopic lentoid bodies. These data suggest that, outside of the normal lens, beta-catenin functions as a coactivator of canonical Wnt signaling to suppress lens fate.     

Nicotine inhibits myofibroblast differentiation in human gingival fibroblasts

Cigarette smoking has been suggested as a risk factor for several periodontal diseases. It has also been found that smokers respond less favorably than non-smokers to periodontal therapy. Previous work in our lab has shown that nicotine inhibits human gingival cell migration. Since myofibroblasts play an important role in wound closure, we asked if nicotine affects gingival wound healing process by regulating myofibroblast differentiation. Human gingival fibroblasts (HGFs) from two patients were cultured in 10% fetal bovine serum cell culture medium. Cells were pretreated with different doses of nicotine (0, 0.01, 0.1, and 1 mM) for 2 h, and then incubated with transforming growth factor beta (TGF-beta1) (0, 0.25, 0.5, and 1 ng/ml) with or without nicotine for 30 h. The expression level of alpha-smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts, was analyzed by Western blots, immunocytochemistry, and real-time polymerase chain reaction (real-time PCR). Phosphorylated p38 mitogen-activated protein kinase (Phospho-p38 MAPK) activity was analyzed by Western blots. TGF-beta1 induced an increase of alpha-SMA protein and mRNA expression, while nicotine (1 mM) inhibited the TGF-beta1-induced expression of alpha-SMA but not beta-actin. Nicotine treatment down-regulated TGF-beta1-induced p38 MAPK phosphorylation. Our results demonstrated for the first time that nicotine inhibits myofibroblast differentiation in human gingival fibroblasts in vitro; supporting the hypothesis that delayed wound healing in smokers may be due to decreased wound contraction by myofibroblasts.     

Gene therapy for tissue regeneration

Tissue repair and regeneration are the normal biological responses of many different tissues in the body to injury. During the healing process, profound changes occur in cell composition and extracellular matrix (ECM) formation. Fibroblasts and equivalent reparative cells migrate to the wounded area and subsequently proliferate. These cells and reparative cells from the surrounding tissue are responsible for the rapid repair which results in tissue regeneration. Growth factors, one of which is transforming growth factor-beta (TGF-beta), stimulate fibroblasts and smooth muscle cells to proliferate and synthesize ECM proteins. This process of early repair provides a rapid way to restore new tissue and mechanical integrity. This early tissue repair process is normally followed by involution, which requires the production and activation of proteases, tissue maturation and remodeling, reorganization and finally regeneration. Alternately, failure to replace the critical components of the ECM, including elastin and basement membrane, results in abnormal regeneration of the epithelial cell layer. Although remodeling should occur during healing, provisional repair may be followed by excessive synthesis and deposition of collagen, which results in irreversible fibrosis and scarring. This excessive fibrosis which occurs in aberrant healing is at least in part mediated by persistent TGF-beta. Because of the central role of collagen in the wound healing process, the pharmacological control of collagen synthesis has been of paramount importance as a possible way to abrogate aberrant healing and prevent irreversible fibrosis. Fibrosis is an abnormal response to tissue injury.     

Expression and potential function of beta-amyloid precursor proteins during cutaneous wound repair

sAPP, the secretory domain of the beta-amyloid precursor protein (APP), exerts a growth promoting and motogenic activity on keratinocytes. Here we report on the expression of APP and its homologue, the amyloid precursor like protein 2 (APLP2), during cutaneous wound repair using a full-thickness excisional wound healing model in mice. In unwounded skin APP was predominantly expressed in the basal cell layer. During wound healing increased suprabasal expression of APP was observed in all cell layers of the hyperproliferative epithelium at the wound margin. APP mRNA was increased up to 2.3-fold, whereas the APLP2 mRNA was decreased. Immunocytochemically, all proliferation competent keratinocytes of the normal as well as the wound site epidermis showed increased expression of APP but not of APLP2. Using culture models of keratinocyte differentiation the release of sAPP was found to be significantly higher in proliferating cells, i.e., when cultured at subconfluency or at low [Ca(2+)], than in quiescent, partially differentiated keratinocytes cultured at confluency or at high [Ca(2+)]. Our results suggest that sAPP secretion is presumably also increased in proliferation competent keratinocytes of the wound margin and that sAPP due to its growth promoting and motogenic function might participate in the control of epidermal wound repair.     

The stabilization of beta-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression

Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.     

CD44 is critically involved in infarct healing by regulating the inflammatory and fibrotic response

Infarct healing is dependent on an inflammatory reaction that results in leukocyte infiltration and clearance of the wound from dead cells and matrix debris. However, optimal infarct healing requires timely activation of "stop signals" that suppress inflammatory mediator synthesis and mediate resolution of the inflammatory infiltrate, promoting formation of a scar. A growing body of evidence suggests that interactions involving the transmembrane receptor CD44 may play an important role in resolution of inflammation and migration of fibroblasts in injured tissues. We examined the role of CD44 signaling in infarct healing and cardiac remodeling using a mouse model of reperfused infarction. CD44 expression was markedly induced in the infarcted myocardium and was localized on infiltrating leukocytes, wound myofibroblasts, and vascular cells. In comparison with wild-type mice, CD44(-/-) animals showed enhanced and prolonged neutrophil and macrophage infiltration and increased expression of proinflammatory cytokines following myocardial infarction. In CD44(null) infarcts, the enhanced inflammatory phase was followed by decreased fibroblast infiltration, reduced collagen deposition, and diminished proliferative activity. Isolated CD44(null) cardiac fibroblasts had reduced proliferation upon stimulation with serum and decreased collagen synthesis in response to TGF-beta in comparison to wild-type fibroblasts. The healing defects in CD44(-/-) mice were associated with enhanced dilative remodeling of the infarcted ventricle, without affecting the size of the infarct. Our findings suggest that CD44-mediated interactions are critically involved in infarct healing. CD44 signaling is important for resolution of the postinfarction inflammatory reaction and regulates fibroblast function.     

Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form

Transforming growth factor-beta (TGF-beta) is a key modulator of epidermal development and homeostasis, and has been shown to potently regulate keratinocyte migration and function during wound repair. There are three cloned TGF-beta receptors termed type I, type II, and type III that are found on most cell types. The types I and II are the signaling receptors, while the type III is believed to facilitate TGF-beta binding to the types I and II receptors. Recently, we reported that in addition to these receptors, human keratinocytes express a 150 kDa TGF-beta 1 binding protein (r150) which forms a heteromeric complex with the TGF-beta signaling receptors. This accessory receptor was described as glycosyl phosphatidylinositol-specific anchored based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). In the present study, we demonstrate that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta 1 with an affinity similar to those of the types I and II TGF-beta signaling receptors. Furthermore, the PIPLC released (soluble) form of this protein is capable of binding TGF-beta 1 independently from the signaling receptors. In addition, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Our observation that r150 interacts with the TGF-beta signaling receptors, together with the finding that the soluble r150 binds TGF-beta 1 suggest that r150 in either its membrane anchored or soluble form may potentiate or antagonize TGF-beta signaling. Elucidating the mechanism by which r150 functions as an accessory molecule in TGF-beta signaling may be critical to understanding the molecular mechanisms underlying the regulation of TGF-beta action in keratinocytes.     

Epithelial Cells Are Active Participants in Vocal Fold Wound Healing: An In Vivo Animal Model of Injury

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.     

NLRC3 deficiency promotes cutaneous wound healing due to the inhibition of p53 signaling

Cutaneous wound healing is a complicated process that is characterized by an initial inflammatory phase followed by a proliferative phase. NLRC3 plays important roles in innate immunity, inflammatory regulation and tumor cell growth. However, the function of NLRC3 in wound healing remains unclear. Here, we investigated the function of NLRC3 in acute cutaneous wound healing using Nlrc3 gene knockout (Nlrc3^?/?) mice. Our results demonstrated that skin wound repair in Nlrc3^?/? mice was significantly accelerated compared with that in wild-type (WT) mice. NLRC3 deficiency promoted the inflammatory and proliferative phases in wounds enhanced the inflammatory response and increased re-epithelialization and granulation tissue formation, and these phenotypes were primarily ascribed to regulatory effects on p53 signaling. Mechanistically, we uncovered novel crosstalk between NLRC3 and p53 signaling and revealed that NLRC3 could mediate the ubiquitination and degradation of p53 in an Hsp90-dependent manner. In conclusion, our study suggests that NLRC3 is a critical negative regulator of the inflammatory response and cell proliferation during wound healing and that blocking NLRC3 may represent a potential approach for accelerating wound healing.     

Keratinocyte differentiation inversely regulates the expression of involucrin and transforming growth factor beta1

Extensive skin loss from a variety of conditions such as severe thermal injury is associated with significant functional morbidity and mortality. In recent years, the healing quality has been improved for patients who suffer burns due in part to the usage of skin replacement mainly prepared from multi-layered sheets of cultured keratinocytes. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-beta1 (TGF-beta1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor and has any functional influence on dermal fibroblasts. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms express different levels of TGF-beta1. To address this hypothesis, keratinocytes were grown in serum free medium (KSFM) supplemented with bovine pituitary extract (50 microg/ml) and EGF (5 microg/ml). When cells reached confluency, conditioned medium was removed and replaced with 50% KSFM with no additives and 50% DMEM without serum and cells were allowed to form multi-layers and differentiate. The conditioned medium was then collected every 48 h up to 24 days and the level of TGF-beta1 and the efficacy of a keratinocyte released fibroblast mitogenic factor were evaluated by ELISA and (3)H-thymidine incorporation, respectively. Northern analysis was also employed to evaluate the expression of TGF-beta1, involucrin, TIMP-1, and 18 S ribosomal RNA in keratinocytes at different times of the onset of differentiation. The microscopic morphology of keratinocytes at different times of induction of cell differentiation showed detachments (nodules) of many regions of keratinocyte sheet from culture substratum within 1-2 weeks. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. The results of TGF-beta1 evaluation revealed that mono-layers of cultured keratinocytes which were round, attached, and proliferating in KSFM + BPE and EGF containing medium released a significantly higher level of TGF-beta1 (196 +/- 58 pg /ml) relative to those grown in multi-layer forms (28 +/- 7.8 pg/ml). A longitudinal experiment was then conducted and the results showed that cells on the onset of differentiation released even greater level of TGF-beta1 (388 +/- 53 pg/ml) relative to those grown in KSFM + BPE and EGF. This finding was consistent with the expression of TGF-beta1 mRNA evaluated in keratinocytes grown in test medium for various duration. In general, the level of TGF-beta1 protein and mRNA gradually reduced to its lowest level within 12 days of growing cells in our test medium. When aliquots of the collected keratinocyte conditioned medium were added to dermal fibroblasts, the level of (3)H-thymidine incorporation increased only in those cells receiving aliquots of conditioned medium containing high levels of TGF-beta1. When involucrin was used as a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. In conclusion, high involucrin expressing differentiated keratinocytes seem to be quiescent in releasing both TGF-beta1 and a fibroblast mitogenic factor.     

A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling

Transforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. Smad2 and Smad3 are direct mediators of TGF-beta signaling, however little is known about the selective activation of Smad2 versus Smad3. The Smad2 and Smad3 knockout mouse phenotypes and studies comparing Smad2 and Smad3 activation of TGF-beta target genes, suggest that Smad2 and Smad3 have distinct roles in TGF-beta signaling. The observation that TGF-beta inhibits proliferation of Smad3-null mammary gland epithelial cells, whereas Smad3 deficient fibroblasts are only partially growth inhibited, suggests that Smad3 has a different role in epithelial cells and fibroblasts. Herein, the current understanding of Smad2 and Smad3-mediated TGF-beta signaling and their relative roles are discussed, in addition to potential mechanisms for the selective activation of Smad2 versus Smad3. Since alterations in the TGF-beta signaling pathway play an important role in promoting tumorigenesis and cancer progression, methods for therapeutic targeting of the TGF-beta signaling pathway are being pursued. Determining how Smad2 or Smad3 differentially regulate the TGF-beta response may translate into developing more effective strategies for cancer therapy.