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.

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides ( In innate immune systems, toll-like receptors (TLRs) 2 are implicated in recognition and signaling of pathogen-associated molecular patterns (1). Stimulation of different TLRs induces distinct patterns of gene expression, which leads to the activation of innate immunity and instructs the development of antigen-specific acquired immunity (2). Among the TLR family, TLR4 plays a critical role in recognition and signaling of bacterial lipopolysaccharide (LPS) (3). TLR4 requires accessory protein MD-2 for an efficient response to LPS (4). We have recently demonstrated the direct interaction between MD-2 and extracellular TLR4 domain (5, 6). MD-2 binds LPS (7), but LPS has been demonstrated to be cross-linked with TLR4 and MD-2 only when coexpressed with CD14 (8), suggesting that LPS is in close proximity to the receptor complex.The lung is constantly challenged by inhaled pathogens, pollutants, and particles that are present in the environment. Pulmonary surfactant, a mixture of lipids and proteins that serves to reduce the surface tension of the alveoli, is involved in the innate immune system of the lung. Recent studies demonstrate that the most abundant component of surfactant protein, surfactant protein A (SP-A), plays important roles in pathogen clearance and inflammatory responses (9 -12). SP-A belongs to the collectin subgroup of the C-type lectin superfamily along with surfactant protein D (SP-D) and mannose-binding lectin. The primary structure of SP-A subunits are composed of a short amino-terminal segment, a collagen-like sequence characterized by Gly-X-Y repeats with an interruption near the midpoint of the domain, a neck domain, and a carbohydrate recognition domain (CRD) (13). Trimeric association occurs by the folding of collagenous domains into triple helices (14) and coiled-coil bundling of ␣-helices in the neck (15). Fully assembled SP-A is a bouquet-like octadecamer consisting of six trimeric subunits that are stabilized by the amino-terminal sequences and disulfide bonds (16).Recent studies from this and other laboratories have demonstrated that SP-A modulates inflammation by interacting with cell surface receptors including CD14 (17), TLR2 (18, 19), signal-inhibitory regulatory protein ␣, and calreticulin/CD91 (20). Although it has been suggested that SP-A activates cellular responses dependent on TLR4 (21), the interactions of SP-A * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan and by Akiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms, chromatin-bound and unbound. The former dissociates from chromatin with progression through the S phase. Recently, we have established a procedure to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM) proteins by immunoprecipitation. In the present study, we applied this procedure to HeLa cells synchronized in each of the G 1 , S, and G 2 /M phases and could detect hMCM heterohexameric complexes in all three. In addition, depending on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously change their phosphorylation states. Concentrating attention on G 2 /M phase hyperphosphorylation, we found hMCM2 and 4 in the complexes to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase was inactivated in temperature-sensitive mutant murine FT210 cells, the G 2 /M hyperphosphorylation of the murine MCM2 and MCM4 and release of the MCMs from chromatin in the G 2 phase were severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell cycle-dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating the MCM complex-chromatin association.The MCM protein family, presently with six members, was originally identified from its involvement in the initiation of DNA replication at autonomously replicating sequences in budding yeast (1-7) and later found to be conserved through eukaryotes (8 -16). Although definite functions of the MCM proteins remain largely unknown, they have been implicated in the regulatory machinery allowing DNA to replicate only once during the S phase (reviewed in Refs. 17 and 18).In mammalian cells, MCM proteins are present in the nuclei in two different forms, one extractable by nonionic detergents and the other tightly associated with chromatin, which is resistant to such extraction. The level of mammalian MCM does not greatly vary during the cell cycle, but the chromatin-bound form gradually becomes dissociated with progression through the S phase (19 -23). It is now assumed that the bound form is associated with prereplicative chromatin and released at the time of replication; the soluble form existing abundantly in G 2 nuclei is considered inactive and no longer capable of binding to chromatin. At least in budding yeast, the chromatin regions to which MCM7 binds during the G 1 phase contain the replication origins (24). However, details of the mode of MCM-chromatin binding remain unclear. In budding yeast and the Xenopus egg extract system, it has been shown that MCM-chromatin binding is regulated through multiple mechanisms, while MCM binds to chromatin depending on CDC6 and the origin recognition complex (24 -27), where the binding is negatively regulated by both S phase and mitotic CDKs (24, 28 -30). However, there is so far no direct evidence as to wheth...

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.