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...

The integrin-associated protein (IAP, CD47) is a 50-kD plasma membrane protein with a single extracellular immunoglobulin variable (IgV)-like domain, a multiply membrane-spanning segment, and alternatively spliced short cytoplasmic tails. On neutrophils, IAP has been shown to function in a signaling complex with β3 integrins. However, the function of IAP on T cells, which express little or no β3 integrin, is not yet defined. Here, we show that mAbs recognizing IAP can enhance proliferation of primary human T cells in the presence of low levels of antiCD3, but have no effect on T cell proliferation on their own. Together with suboptimal concentrations of anti-CD3, engagement of IAP also enhances IL-2 production in Jurkat cells, an apparently integrin-independent function of IAP. Nonetheless, costimulation by IAP ligation requires cell adhesion. IAP costimulation does not require CD28. Furthermore, anti-IAP, but not anti-CD28, synergizes with suboptimal anti-CD3 to enhance tyrosine phosphorylation of the CD3 ζ chain and the T cell–specific tyrosine kinase Zap70. Ligation of human IAP transfected into the hemoglobin-specific 3.L2 murine T cell hybridoma costimulates activation for IL-2 secretion both with anti-CD3 and with antigenic peptides on antigen-presenting cells (APCs). Moreover, ligation of IAP but not CD28 can convert antagonist peptides into agonists in 3.L2 cells. Using costimulation by IAP ligation as an assay to analyze the structure–function relationships in IAP signaling, we find that both the extracellular and multiply membrane-spanning domains of IAP are necessary for synergy with the antigen receptor, but the alternatively spliced cytoplasmic tails are not. These data demonstrate that IAP ligation initiates an adhesiondependent costimulatory pathway distinct from CD28. We hypothesize that anti-IAP generates the costimulatory signal because it modulates interactions of the IgV domain with other plasma membrane molecules; this in turn activates effector functions of the multiply membrane-spanning domain of IAP. This model may have general significance for how IAP functions in cell activation.

Abstract. Integrin-associated protein (IAP/CD47) is physically associated with the e~v133 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg-Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-lAP antibodies also inhibit binding of Vn-coated particles to many cells expressing av[33. However, prior studies with anti-lAP did not directly address IAP function because they could not distinguish between IAP blockade and antibody-induced signaling effects on cells. To better determine the function of lAP, we have characterized and used an IAPdeficient human cell line. Despite expressing av integrins, these cells do not bind Vn-coated particles unless transfected with IAP expression constructs. Increasing the level of av[33 expression or increasing Vn density on the particle does not overcome the requirement for IAP. All known splice variants of IAP restore Vn particle binding equivalently. Indeed, the membraneanchored IAP Ig variable domain suffices to mediate Vn particle binding in this system, while the multiply membrane-spanning and cytoplasmic domains are dispensable. In all cases, adhesion to a Vn-coated surface and fibronectin particle binding through e~5131 fibronectin receptors are independent of IAP expression. These data demonstrate that some av integrin ligand-binding functions are IAP independent, whereas others require lAP, presumably through direct physical interaction between its Ig domain and the integrin.I NTE6RINS are heterodimeric cell surface receptors that mediate binding to other cells and to ligands in the extracellular matrix. Integrin-dependent ligand binding and clustering-induced signal transduction lead to changes in cell morphology and activation state. Conversely, signals inside the cell can result in changes in integrin ligand affinity and avidity. One of the proteins implicated in these regulatory events is integrin-associated protein (IAP). 1 IAP is a unique Ig family member with an amino-terminal Ig variable (IgV) domain, a multiply membrane-spanning domain, and a short alternatively spliced carboxy-ter-

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...