Secreted frizzled related proteins (sFRPs) have emerged as key regulators of a wide range of developmental and disease processes, with virtually all known functions of mammalian sFRPs attributed to their ability to antagonize Wnt signaling. Recently however, the Xenopus and zebrafish sFRP, Sizzled, was shown to function as an antagonist of Chordin processing by Tolloid-like metalloproteinases, leading to the proposal that sFRPs may function as evolutionarily-conserved antagonists of the chordinase activities of this class of proteinases. Herein, in contrast to this proposal, we show that the mammalian sFRP, sFRP2, does not affect Chordin processing, but instead can serve as a direct enhancer of the procollagen C-proteinase activity of Tolloid-like metalloproteinases. We further show that the level of fibrosis, in which procollagen processing by Tolloid-like proteinases plays a rate-limiting role, is markedly reduced in sFRP2-null mice subjected to myocardial infarction. Importantly, this reduced level of fibrosis is accompanied by significantly improved cardiac function. This study thus uncovers a novel function for sFRP2 and a potential therapeutic application for sFRP2 antagonism in controlling fibrosis in the infarcted heart.

Retrovirus‐mediated gene transfer of the human beta‐globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta‐globin gene disorders. However, expression of the transduced beta‐globin gene linked to its proximal cis‐acting sequences (‐0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta‐locus control region (beta‐LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta‐like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta‐globin gene, but initial attempts to incorporate beta‐LCR derivatives in retroviral vectors resulted in the production of low‐titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta‐globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow‐repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta‐LCR derivatives in dimethylsulfoxide‐induced murine erythroleukemia cells and to achieve ratios of human beta‐globin/murine beta maj‐globin mRNA, on a per gene basis, as high as 80%.

Bone morphogenetic protein-1-like proteinases play key roles in formation of the extracellular matrix (ECM) in vertebrates via biosynthetic processing of precursors into mature functional proteins involved in ECM assembly. Such processing includes proteolytic activation of the zymogen for lysyl oxidase. Fibronectin (FN) is an abundant protein component of the ECM that is capable of regulating manifold cellular functions through its interactions with various ECM and cell surface proteins. It was previously shown that proteolytic activation of lysyl oxidase is much reduced in cultures of FN-null mouse embryo fibroblasts (MEFs). Here we demonstrate that cellular fibronectin, the form produced by fibroblasts and various other tissue cell types, and plasma fibronectin bind BMP1 with dissociation constants (K D ) of ϳ100 nM, consistent with a physiological role. Also consistent with such a role, cellular fibronectin FN is shown to positively regulate BMP1 processing activity against Chordin, probiglycan, and type I procollagen in vitro. Endogenous FN and BMP1 are demonstrated to co-localize in cell layers and to form complexes in culture medium. In addition, processing of endogenous BMP1 substrates Chordin, probiglycan, and procollagen is demonstrated to be strikingly reduced in cultures of FN ؊/؊ MEFs compared with FN ؉/؊ MEF cultures despite similar levels of endogenous BMP1. These data support the conclusion that FN binds BMP1-like proteinases in vivo and that FN is an important determinant of the in vivo activity levels of BMP1-like proteinases. Fibronectin (FN)3 is a noncollagenous extracellular matrix (ECM) glycoprotein of relatively high abundance that regulates a wide variety of cellular functions, including adhesion, migration, proliferation, differentiation, and apoptosis (1-4). FN is secreted as a disulfide-bonded dimer, and each subunit comprises 12 type I, 2 type II, and 15-17 type III FN modules as well as a "variable" (V) region that lacks homology to other protein domains (3). FN is found as two different major forms, plasma fibronectin (pFN), a soluble form synthesized by hepatocytes, and cellular fibronectin (cFN), which is locally expressed by many other cell types in various tissues (5). Both forms can be assembled into a fibrillar ECM by cultured fibroblasts (6). Differences between cFN and pFN arise from alternative RNA splicing in three regions; two type III repeats (designated EDA and EDB) and the V region. EDA and EDB are present in cFN but absent from pFN, whereas although only one subunit of the pFN dimer contains the V region, almost all cFN subunits contain this region (7). These differences in domain structure contribute to distinct functions for pFN and cFN; cFN plays roles in the dynamic tissue modeling of early embryogenesis and wound healing (8), whereas pFN subserves roles in hemostasis and thrombosis and immune responses (3, 9 -11) and provides a reservoir for deposition in tissue (12).BMP1-like proteinases are evolutionary conserved extracellular metalloproteinases that play multip...

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