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Activation of transforming growth factor ␤ receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the ␦EF1/ Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like ␦EF1, SIP1 binds to 5-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2 promoter in vitro, prevented expression of the endogenous Xbra gene in early Xenopus embryos. Therefore, SIP1, like ␦EF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor ␤ members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo.Ligands of the TGF-␤ 1 family exert their biological effects by activating serine/threonine kinase receptor complexes, which in turn activate intracellular mediators, the Smad proteins. Smads were initially identified by means of genetic studies in Drosophila and Caenorhabditis elegans as Mad and Sma gene products, respectively. Nine different vertebrate Smads have been isolated (reviewed in Refs. 1-3; Ref. 4). These proteins are characterized by a three-domain structure containing conserved N-terminal and C-terminal domains, called the MH1 and MH2 domains, which flank a more variable, proline-rich linker region. The Smads can be classified into three subgroups based on their distinct functions. The receptor-regulated Smads (Smad1, 2, 3, 5, and 8) contain a conserved SSXS motif at their extreme C-terminal end. Upon ligand stimulation, two serines in this motif are directly phosphorylated by specific type I receptors. Once activated, these Smads associate with Smad4, a common mediator Smad, and the heteromeric complexes translocate to the nucleus where they mediate responses to specific ligands. Smads 1, 5, and 8 act in bone morphogenetic protein (BMP) pathways, whereas Smads 2 and 3 act in activin and TGF-␤ pathways. A third group of Smads, the inhibitory Smads (Smad6 and Smad7), prevent the activation of receptorregulated Smads or their heteromerization with Smad4. Functional homologues of inhibitory Smads and the common mediator Smad in Drosophila have been identified as Dad and Medea, respectively (1-3).In the absence of signaling, Smads are kept in a latent conformation through an intramolecular interaction between the MH1 and MH2 domains. Activation of receptor-regulated Smads has...

Objective. Ligands and antagonists of the WNT pathway are linked to osteoporosis and osteoarthritis. In particular, polymorphisms in the FRZB gene, a secreted WNT antagonist, have been associated with osteoarthritis. The aim of this study was to examine cartilage and bone in Frzb ؊/؊ mice. Methods. The Frzb gene in mice was inactivated using a Cre/loxP strategy. Three models of osteoarthritis were used: collagenase, papain, and methylated bovine serum albumin induced. Bone biology was studied using density measurements and microfocal computed tomography. Bone stiffness and mechanical loading-induced bone adaptation were studied by compression of the ulnae.Results. Targeted deletion of the Frzb gene in mice increased articular cartilage loss during arthritis triggered by instability, enzymatic injury, or inflammation. Cartilage damage in Frzb ؊/؊ mice was associated with increased WNT signaling and matrix metalloproteinase 3 (MMP-3) expression and activity. Frzb ؊/؊ mice had increased cortical bone thickness and density, resulting in stiffer bones, as demonstrated by stressstrain relationship analyses. Moreover, Frzb ؊/؊ mice had an increased periosteal anabolic response to mechanical loading as compared with wild-type mice.Conclusion. The genetic association between osteoarthritis and FRZB polymorphisms is corroborated by increased cartilage proteoglycan loss in 3 different models of arthritis in Frzb ؊/؊ mice. Loss of Frzb may contribute to cartilage damage by increasing the expression and activity of MMPs, in a WNT-dependent and WNT-independent manner. FRZB deficiency also resulted in thicker cortical bone, with increased stiffness and higher cortical appositional bone formation after loading. This may contribute to the development of osteoarthritis by producing increased strain on the articular cartilage during normal locomotion but may protect against osteoporotic fractures.Osteoarthritis and osteoporosis are common joint and bone diseases that cause significant morbidity and disability in the aging population. Osteoarthritis is primarily characterized by degeneration of the articular cartilage and leads to loss of joint function, and patients often require surgery for placement of a prosthesis to correct it (1). Drugs that convincingly affect the disease process beyond pain relief are not yet available. Osteoporosis is defined by decreased cortical and trabecular bone density and typically results in hip and vertebral fractures (2). Current antiosteoporosis agents inhibit osteoclast-driven bone resorption or stimulate osteoblast-driven bone synthesis, but their long-term use can cause drug safety problems (2). Clinical observations suggest that there is an inverse relationship between osteoarthritis and osteoporosis (3), but this hypothesis remains controversial, particularly since it is not supported by a known molecular mechanism.A role of ligands and antagonists of the WNT

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The human extracellular matrix protein 1 (Ecm1) gene is located at chromosome band 1q21 close to the epidermal differentiation complex and is transcribed in two discrete mRNAs: a full length Ecm1a and a shorter, alternatively spliced, Ecm1b transcript, the expression of which is restricted to tonsils and skin. The chromosomal localization and the Ecm1b expression in skin prompted us to investigate the role of Ecm1 in keratinocyte differentiation. In this study, we provide evidence for the existence of a relationship between keratinocyte differentiation and expression of the Ecm1b transcript. Cultures of subconfluent undifferentiated normal human keratinocytes express only Ecm1a. Upon reaching confluence, the cells start to differentiate, as measured by keratin K10 mRNA expression. Concomitantly Ecm1b mRNA expression is induced, although expression of Ecm1a mRNA remains unchanged. In addition, treatment of undifferentiated normal human keratinocyte cells with 12-O-tetradecanoyl-phorbol-13-acetate strongly induces the expression of Ecm1b mRNA. Expression of Ecm1b can also be induced by coculturing normal human keratinocytes with lethally irradiated feeder cells and by a diffusible factor secreted by stromal cells. In adult human skin, Ecm1a mRNA is expressed throughout the epidermis with the strongest expression in the basal and first suprabasal cell layers, whereas expression of Ecm1b mRNA is predominantly found in spinous and granular cell layers. Immunohistochemically, Ecm1a expression is almost completely restricted to the basal cell layer, whereas Ecm1b is detected in the suprabasal layers. These results are strongly suggestive of a role for Ecm1b in terminal keratinocyte differentiation, which is also supported by the localization of the Ecm1 gene at 1q21. Refinement of its genomic localization, however, placed Ecm1 centromeric of the epidermal differentiation complex.