Mutations in fibroblast growth factor (FGF) receptor 3 lead to the human dwarfism syndrome achondroplasia. Using a limb culture system, we have analyzed the role of FGF signaling and its interaction with the Ihh/Pthlh and BMP pathways in regulating chondrocyte differentiation. In contrast to previous suggestions, we demonstrate that FGF signaling accelerates both the onset and the pace of hypertrophic differentiation. We furthermore found that FGF and BMP signaling act in an antagonistic relationship regulating chondrocyte proliferation, Ihh expression, and the process of hypertrophic differentiation. Importantly, BMP signaling rescues the reduced domains of proliferating and hypertrophic chondrocytes in a mouse model for achondroplasia. We propose a model in which the balance of BMP and FGF signaling adjusts the pace of the differentiation process to the proliferation rate.

The longitudinal growth of the skeleton arises from the continuous process of endochondral ossification occurring at the ends of growing long bones. Dwarfism results when this process is disrupted, as in the autosomal dominant human skeletal diseases hypochondroplasia (HCH), achondroplasia (ACH) and thanatophoric dysplasia (TD). Interestingly, these disorders display a graded spectrum of phenotypic severity and are the result of distinct missense mutations in the fibroblast growth factor receptor 3 gene (FGFR3). TD, characterized by neonatal lethality and profound dwarfism, is the result of FGFR3 mutations, including an R248C substitution in the extracellular domain or a K650E substitution in the tyrosine kinase (TK) domain. ACH, which is non-lethal and presents less severe dwarfism, results almost exclusively from a G380R substitution in the transmembrane domain. Homozygous achondroplasia resembles the phenotype of TD. In this report the effect of the ACH and TD mutations on the activity and regulation of FGFR3 are analysed. We showed that each of the mutations constitutively activate the receptor, as evidenced by ligand-independent receptor tyrosine phosphorylation and cell proliferation. Moreover, the mutations that are responsible for TD were more strongly activating than the mutation causing ACH, providing a biochemical explanation for the observation that the phenotype of TD is more severe than that of ACH.

Wnt signaling is essential for many developmental processes, including skeletogenesis. To investigate the effects of Wnt signaling during skeletogenesis we studied the effects of Wnt on cultured chondrocytic cells and differentiating limb-bud mesenchyme. We showed that Wnt3a strongly repressed chondrogenesis and chondrocyte gene expression. Canonical Wnt signaling was responsible for the repression of differentiation, as evidenced by results showing that inhibition of glycogen synthase kinase 3 or expression of ␤-catenin caused similar repression of differentiation. Significantly, we showed that the transcription repressor Twist1 is induced by canonical Wnt signaling. Expression of Twist1 strongly inhibited chondrocyte gene expression and short hairpin RNA knockdown of Twist1 transcript levels caused increased expression of the chondrocyte-specific genes aggrecan and type II collagen. Interestingly, Twist1 interfered with BMP2-induced expression of aggrecan and type II collagen expression and knockdown of Twist1 augmented BMP2-induced aggrecan and type II collagen expression. These data support the conclusions that Twist1 contributes to the repression of chondrogenesis and chondrocyte gene expression resulting from canonical Wnt signaling and that Twist1 interferes with BMP-dependent signaling.Wnt signaling is divided into canonical and non-canonical pathways. Canonical Wnt signaling regulates the protein levels of ␤-catenin (␤cat).2 The binding of Wnt ligands to cell-surface receptors leads to the activation of the intracellular protein Dishevelled. When activated, Dishevelled is released from the receptor complex at the cell surface and interacts with the multiprotein complex that controls ␤cat levels. This complex includes axin, the adenomatous polyposis coli gene product, glycogen synthase kinase 3 (GSK3), ␤cat, and other proteins (1). Within the complex, phosphorylation of ␤cat by GSK3 leads to its ubiquitination and degradation by the proteasome. GSK3 is inhibited by activated Dishevelled. This in turn stabilizes ␤cat, thereby increasing the amount of ␤cat, which accumulates in the nucleus and regulates gene expression in conjunction with the TCF/Lef family of transcription factors. Thus the canonical Wnt signaling pathway is in part a transcription control pathway that regulates the levels of the transcription co-activator ␤cat. Non-canonical Wnt signaling pathways are subdivided into the Wntcalcium and planar cell polarity pathways (2-4). The Wnt-calcium pathway increases intracellular calcium levels in response to Wnt signaling. Like canonical Wnt signaling, this pathway also requires Dishevelled (5). The release of calcium stimulates calcium-dependent kinases and transcription factors, like the nuclear factor of activated T-cells family of transcription factors (6, 7). The planar cell polarity pathway, also linked to Dishevelled activation, requires the Rho family GTPases and Jun kinase. Through this pathway Wnt signaling regulates polarized cell movements.A hierarchy of Wnt signaling regulates diverse...

We studied autocrine transforming growth factor (TGF)beta signaling in kidney epithelium. Cultured proximal tubule cells showed regulated signaling that was high during log-phase growth, low during contact-inhibited differentiation, and rapidly increased during regeneration of wounded epithelium. Autoregulation of signaling correlated with TGFbeta receptor and Smad7 levels, but not with active TGFbeta, which was barely measurable in the growth medium. Confluent differentiated cells with low receptor and high Smad7 levels exhibited blunted responses to saturating concentrations of exogenously provided active TGFbeta, suggesting that TGFbeta signaling homeostasis was achieved by cell density-dependent modulation of signaling intermediates. Antagonism of Alk5 kinase, the TGFbeta type I receptor, dramatically accelerated the induction of differentiation in sparse, proliferating cultures and permitted better retention of differentiated features in regenerating cells of wounded, confluent cultures. Alk5 antagonism accelerated the differentiation of cells in proximal tubule primary cultures while simultaneously increasing their proliferation. Consequently, Alk5-inhibited primary cultures formed confluent, differentiated monolayers faster than untreated cultures. Furthermore, treatment with an Alk5 antagonist promoted kidney repair reflected by increased tubule differentiation and decreased tubulo-interstitial pathology during the recovery phase following ischemic injury in vivo. Our results show that autocrine TGFbeta signaling in proliferating proximal tubule cells exceeds the levels that are necessary for physiological regeneration. To that end, TGFbeta signaling is redundant and maladaptive during tubule repair by epithelial regeneration.

Canonical Wnt signaling is clearly required for skeletal development and bone formation. However, the targets of Wnt signaling that convert this signal into bone are unclear. Identification of these targets will yield insight into normal bone physiology and suggest new therapeutics for treatment of bone disease. Here we show that an essential regulator of bone development, FGF18, is a direct target of canonical Wnt signaling. A single DNA binding site for the Wnt-dependent transcription factors TCF/Lef accounted for the stimulation of the fgf18 promoter in response to Wnt signaling. Additionally, targeted disruption of ␤cat blocked fgf18 expression in vivo. Partially overlapping the TCF/Lef binding site is a Runx2 binding site and experiments showed that Runx2 and TCF/Lef work cooperatively to induce fgf18 expression. RNA interference knockdown of Runx2 inhibited and Runx2 forced expression augmented the induction of fgf18 by canonical Wnt signaling. Significantly, Runx2 formed a complex with Lef1 or TCF4 and this complex bound the composite binding site in the fgf18 promoter. These results demonstrate that two transcription pathways that are essential for bone, physically and functionally converge at the fgf18 promoter.

The vertebrate skeleton develops from a template that is initially constructed of cartilage. The formation of this template is controlled by a series of interdependent steps that supply spatial information and morphogenic cues that direct mesenchymal cell condensation and differentiation in a process known as chondrogenesis. Mesenchymal cells derived from the lateral plate mesoderm condense to form the rudiments of the appendicular skeleton. The processes that control the condensation of mesenchyme are poorly understood. Cell-cell adhesion certainly contributes to mesenchymal cell condensation as demonstrated in studies showing that homotypic interactions of N-cadherin are required during chondrogenesis (1, 2). In addition, a number of extracellular signaling molecules have been proposed to regulate chondrogenesis by regulating both condensation and differentiation. WNTs (3), fibroblast growth factors (4), sonic hedgehog (5), and bone morphogenetic proteins (BMPs) 1 (6 -8) all contribute to chondrogenesis. Increasing evidence indicates that BMPs have a central role in this process. For example, loss of function experiments using dominant negative BMP receptors suppresses chondrogenesis during chicken limb development (9, 10). Conversely, overexpression of BMPs or activated BMP receptors dramatically augments chondrogenesis during limb development (10, 11). These data suggest that elucidating pathways that control BMP expression will be vital to understanding chondrogenesis.Intracellular calcium ([Ca 2ϩ ] i ) is a universal intracellular signal that intersects with many signaling molecules to regulate gene expression (12). However, the role of intracellular calcium in chondrogenesis is virtually uncharacterized. Elevations of [Ca 2ϩ ] i activate a number of signaling cascades, including the calcineurin/nuclear factor of activated T-cell (NFAT) pathway (13). Prolonged elevations of [Ca 2ϩ ] i activate the phosphatase calcineurin, through interaction with Ca 2ϩ :calmodulin (14). In turn, calcineurin dephosphorylates a set of substrates, including the transcription factors, NFATs (15). Dephosphorylation of NFATs results in translocation of the protein from the cytoplasm to the nucleus (16). In the nucleus, NFATs activate gene expression in cooperation with other transcriptional regulators (17, 18). NFATs are a family of four transcription factors, NFAT1(p, c2), NFAT2(c, c1), NFAT3, and NFAT4(x,c3). Although well known for their ability to regulate cytokine gene expression in immune cells (15), NFATs are broadly expressed and have essential functions outside of the immune system (18 -21). The transcriptional targets of NFATs outside of immune cells, however, are incompletely characterized. Here we report that elevation of [Ca 2ϩ ] i induce chondrogenesis using a pathway requiring calcineurin, NFAT4, and BMP expression. In this pathway BMP2 expression is induced by activated calcineurin/NFAT. Subsequently, BMP2 induces chondrogenesis through an autocrine loop. were generously provided by Jane Aubin. Cells wer...

Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.

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