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Mice with disruption of Lrrk1 and patients with nonfunctional mutant Lrrk1 exhibit severe osteopetrosis phenotypes because of osteoclast cytoskeletal dysfunction. To understand how Lrrk1 regulates osteoclast function by modulating cytoskeleton rearrangement, we examined the proteins that are differentially phosphorylated in wild‐type mice and Lrrk1‐deficient osteoclasts by metal affinity purification coupled liquid chromatography/mass spectrometry (LC/MS) analyses. One of the candidates that we identified by LC/MS is L‐plastin, an actin bundling protein. We found that phosphorylation of L‐plastin at serine (Ser) residues 5 was present in wild‐type osteoclasts but not in Lrrk1‐deficient cells. Western blot analyses with antibodies specific for Ser5 phosphorylated L‐plastin confirmed the reduced L‐plastin Ser5 phosphorylation in Lrrk1 knockout (KO) osteoclasts. micro computed tomography (Micro‐CT) analyses revealed that the trabecular bone volume of the distal femur was increased by 27% in the 16 to 21‐week‐old L‐plastin KO females as compared with the wild‐type control mice. The ratio of bone volume to tissue volume and connectivity density were increased by 44% and 47% (both P

Multiple sclerosis (MS) is caused by immune-mediated damage of myelin sheath. Current therapies aim to block such immune responses. However, this blocking is not sufficiently specific and hence compromises immunity, leading to severe side effects. In addition, blocking medications usually provide transient effects and require frequent administration, which further increases the chance to compromise immunity. In this regard, myelin-specific therapy may provide the desired specificity and a long-lasting therapeutic effect by inducing myelin-specific regulatory T (Treg) cells. Tolerogenic dendritic cells (TolDCs) are one such therapy. However, ex vivo generated TolDCs may be converted into immunogenic DCs in a proinflammatory environment. In this study, we identified a potential novel myelin-specific therapy that works with immunogenic DCs, hence without the in vivo conversion concern. We showed that immunization with DCs, engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase for de novo synthesis of a focally high 1,25-dihydroxyvitamin D concentration in the peripheral lymphoid tissues, induced Treg cells. In addition, such engineered DCs, when pulsed with a myelin antigen, led to myelin-specific suppression of ongoing experimental allergic encephalomyelitis (an MS animal model), and the disease suppression depended on forkhead-box-protein-P3(foxp3)+ Treg cells. Our data support a novel concept that immunogenic DCs can be engineered for myelin-specific therapy for MS.—Li, C.-H., Zhang, J., Baylink, D. J., Wang, X., Goparaju, N. B., Xu, Y., Wasnik, S., Cheng, Y., Berumen, E. C., Qin, X., Lau, K.-H. W., Tang, X. Dendritic cells, engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase and pulsed with a myelin antigen, provide myelin-specific suppression of ongoing experimental allergic encephalomyelitis.

Cyclooxygenase 2 (COX-2) is essential for normal tissue repair. Although COX-2 is known to enhance the differentiation of mesenchymal stem cells (MSCs), how COX-2 regulates MSC differentiation into different tissue-specific progenitors to promote tissue repair remains unknown. Because it has been shown that COX-2 is critical for normal bone repair and local COX-2 overexpression in fracture sites accelerates fracture repair, this study aimed to determine the MSC subsets that are targeted by COX-2. We showed that CD90+ mouse skeletal stem cells (mSSCs; i.e., CD45−Tie2−AlphaV+ MSCs) were selectively recruited by macrophage/monocyte chemoattractant protein 1 into fracture sites following local COX-2 overexpression. In addition, local COX-2 overexpression augmented osteoblast differentiation and suppressed chondrocyte differentiation in CD90+ mSSCs, which depended on canonical WNT signaling. CD90 depletion data demonstrated that local COX-2 overexpression targeted CD90+ mSSCs to accelerate fracture repair. In conclusion, CD90+ mSSCs are promising targets for the acceleration of bone repair.

Current therapies for gut inflammation have not reached the desired specificity and are attended by unintended immune suppression. This study aimed to provide evidence for supporting a hypothesis that direct in vivo augmentation of the induction of gut-homing regulatory T (Treg) cells is a strategy of expected specificity for the treatment of chronic intestinal inflammation (e.g., inflammatory bowel disease). We showed that dendritic cells (DCs), engineered to de novo produce high concentrations of both 1,25-dihydroxyvitamin D, the active vitamin D metabolite, and retinoic acid, an active vitamin A metabolite, augmented the induction of T cells that express both the regulatory molecule Foxp3 and the gut-homing receptor CCR9 in vitro and in vivo. In vivo, the newly generated Ag-specific Foxp3+ T cells homed to intestines. Additionally, transfer of such engineered DCs robustly suppressed ongoing experimental colitis. Moreover, CD4+ T cells from spleens of the mice transferred with the engineered DCs suppressed experimental colitis in syngeneic hosts. The data suggest that the engineered DCs enhance regulatory function in CD4+ T cell population in peripheral lymphoid tissues. Finally, we showed that colitis suppression following in vivo transfer of the engineered DCs was significantly reduced when Foxp3+ Treg cells were depleted. The data indicate that maximal colitis suppression mediated by the engineered DCs requires Treg cells. Collectively, our data support that DCs de novo overproducing both 1,25-dihydroxyvitamin D and retinoic acid are a promising novel therapy for chronic intestinal inflammation.

This study identifies that cyclooxygenase 2 targets CD90+ skeletal stem cells in fracture sites to accelerate fracture repair., Cyclooxygenase 2 (COX-2) is essential for normal tissue repair. Although COX-2 is known to enhance the differentiation of mesenchymal stem cells (MSCs), how COX-2 regulates MSC differentiation into different tissue-specific progenitors to promote tissue repair remains unknown. Because it has been shown that COX-2 is critical for normal bone repair and local COX-2 overexpression in fracture sites accelerates fracture repair, this study aimed to determine the MSC subsets that are targeted by COX-2. We showed that CD90+ mouse skeletal stem cells (mSSCs; i.e., CD45−Tie2−AlphaV+ MSCs) were selectively recruited by macrophage/monocyte chemoattractant protein 1 into fracture sites following local COX-2 overexpression. In addition, local COX-2 overexpression augmented osteoblast differentiation and suppressed chondrocyte differentiation in CD90+ mSSCs, which depended on canonical WNT signaling. CD90 depletion data demonstrated that local COX-2 overexpression targeted CD90+ mSSCs to accelerate fracture repair. In conclusion, CD90+ mSSCs are promising targets for the acceleration of bone repair.

An indispensable role of macrophages in bone repair has been well recognized. Previous data have demonstrated the copresence of M1 macrophages and mesenchymal stem cells (MSCs) during the proinflammatory stage of bone repair. However, the exact role of M1 macrophages in MSC function and bone repair is unknown. This study aimed to define the role of M1 macrophages at bone injury sites via the function of 1,25-Dihydroxyvitamin D (1,25[OH]2D) in suppressing M1 but promoting M2 differentiation. We showed that 1,25(OH)2D suppressed M1 macrophage–mediated enhancement of MSC migration. Additionally, 1,25(OH)2D inhibited M1 macrophage secretion of osteogenic proteins (i.e., Oncostatin M, TNF-α, and IL-6). Importantly, the 1,25(OH)2D-mediated suppression of osteogenic function in M1 macrophages at the proinflammatory stage was associated with 1,25(OH)2D-mediated reduction of MSC abundance, compromised osteogenic potential of MSCs, and impairment of fracture repair. Furthermore, outside the proinflammatory stage, 1,25(OH)2D treatment did not suppress fracture repair. Accordingly, our data support 2 conclusions: (a) M1 macrophages are important for the recruitment and osteogenic priming of MSCs and, hence, are necessary for fracture repair, and (b) under vitamin D–sufficient conditions, 1,25(OH)2D treatment is unnecessary and can be detrimental if provided during the proinflammatory stage of fracture healing.

In the past there have been a multitude of studies that ardently support the role of arginase II (Arg II) in vascular and endothelial disorders; however, the regulation and function of Arg II in autoimmune diseases has thus far remained unclear. Here we report that a global Arg II null mutation in mice suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. During EAE, both Arg I and Arg II were induced in spinal cords, but only Arg II was induced in spleens and splenic dendritic cells (DCs). DC activation by lipopolysaccharide (LPS), CD40L or TLR8 agonist significantly enhanced Arg II expression without affecting Arg I expression. Conversely, DC differentiating cytokines [IL‐4 and granulocyte macrophage‐colony‐stimulating factor (GM‐CSF)] yielded opposite effects. In addition, Arg I and Arg II were regulated differentially during Th1 and Th17 cell polarization. Arg II deficiency in mice delayed EAE onset, ameliorated clinical symptoms and reduced myelin loss, accompanied by a remarkable reduction in the EAE‐induced spinal cord expression of Th17 cell markers (IL‐17 and ROR γt). The abundance of Th17 cells and IL‐23(+) cells in relevant draining lymph nodes was significantly reduced in Arg II knockout mice. In activated DCs, Arg II deficiency significantly suppressed the expression of Th17‐differentiating cytokines IL‐23 and IL‐6. Interestingly, Arg II deficiency did not lead to any compensatory increase in Arg I expression in vivo and in vitro. In conclusion, Arg II was identified as a factor promoting EAE likely via an Arg I‐independent mechanism. Arg II may promote EAE by enhancing DC production of Th17‐differentiating cytokines. Specific inhibition of Arg II could be a potential therapy for multiple sclerosis.

Systemic 1,25(OH)2D3 treatment ameliorating murine inflammatory bowel diseases (IBD) could not be applied to patients because of hypercalcemia. We tested the hypothesis that increasing 1,25(OH)2D3 synthesis locally by targeting delivery of the 1α-hydroxylase gene (CYP27B1) to the inflamed bowel would ameliorate IBD without causing hypercalcemia. Our targeting strategy is the use of CD11b(+)/Gr1(+) monocytes as the cell vehicle and a macrophage-specific promoter (Mac1) to control CYP27B1 expression. The CD11b(+)/Gr1(+) monocytes migrated initially to inflamed colon and some healthy tissues in dextran sulfate sodium (DSS) colitis mice; however, only the migration of monocytes to the inflamed colon was sustained. Adoptive transfer of Gr1(+) monocytes did not cause hepatic injury. Infusion of Mac1-CYP27B1-modified monocytes increased body weight gain, survival, and colon length, and expedited mucosal regeneration. Expression of pathogenic Th17 and Th1 cytokines (interleukin (IL)-17a and interferon (IFN)-α) was decreased, while expression of protective Th2 cytokines (IL-5 and IL-13) was increased, by the treatment. This therapy also enhanced tight junction gene expression in the colon. No hypercalcemia occurred following this therapy. In conclusion, we have for the first time obtained proof-of-principle evidence for a novel monocyte-based adoptive CYP27B1 gene therapy using a mouse IBD model. This strategy could be developed into a novel therapy for IBD and other autoimmune diseases.

Qa-1 epitopes, the peptides that bind to non-classical major histocompatibility complex Ib Qa-1 molecules and are recognized by Qa-1-restricted CD8+ regulatory T (Treg) cells, have been identified in pathogenic autoimmune cells that attack myelin sheath in experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis [MS]). Additionally, immunization with such epitopes ameliorates the EAE. However, identification of such epitopes requires knowledge of the pathogenic autoimmune cells which are largely unknown in MS patients. Hence, we asked whether the CD8+ Treg cells could directly target the myelin sheath to ameliorate EAE. To address this question, we analyzed Qa-1 epitopes in myelin oligodendrocyte glycoprotein (MOG that is a protein in myelin sheath). Here, we report identification of a MOG-specific Qa-1 epitope. Immunization with this epitope suppressed ongoing EAE, which was abrogated by CD8+ T cell depletion. Additionally, the epitope immunization activated the epitope-specific CD8+ T cells which specifically accumulated in the CNS-draining cervical lymph nodes. Finally, CD8+ T cells primed by the epitope immunization transferred EAE suppression. Hence, this study reveals a novel regulatory mechanism mediated by the CD8+ Treg cells. We propose that immunization with myelin-specific HLA-E epitopes (human homologues of Qa-1 epitopes) is a promising therapy for MS.

Multiple sclerosis (MS) is an incurable central nervous system autoimmune disease. Understanding MS pathogenesis is essential for the development of new MS therapies. In the present study, we identified a novel microRNA (miR) that regulates experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression of miR223 was up-regulated specifically in spinal cords and lymphoid organs but not in other examined tissues. A global miR223 knockout (miR223(-/-) ) in mice led to a significant delay in EAE onset, reduction in spinal cord lesion, and lessening of neurological symptoms. These protective effects could be reproduced in bone marrow chimeras reconstituted with miR223(-/-) haematopoietic stem cells. We also found that miR223 deficiency reduced T helper type 1 (Th1) and Th17 infiltration into spinal cords. To address underlying mechanisms, we investigated the role of miR223 in regulating the function, development and interaction of the major immune cells. Expression of the genes associated with dendritic cell (DC) activation (CD86 and MHC II) and Th1 and Th17 differentiation [interleukin-12 (IL-12) and IL-23, respectively] was significantly decreased in the spleens of miR223(-/-) mice bearing EAE. The miR223(-/-) DCs expressed significantly lower levels of basal and lipopolysaccharide-induced IL-12 and IL-23 compared with the wild-type DCs. These data are consistent with the observed lower efficiency of miR223(-/-) DCs to support Th1 and Th17 differentiation from naive T cells over-expressing an EAE antigen-specific T-cell receptor. Our data suggest that miR223 promotes EAE, probably through enhancing DC activation and subsequently the differentiation of naive T cells toward Th1 and Th17 effector cells.

Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser144, catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains.

We have investigated the effect of tumor necrosis factor-alpha (TNF-alpha) on the production of extracellular matrix-degrading proteases in skeletal muscles. Using microarray, quantitative PCR, Western blotting, and zymography, we found that TNF-alpha drastically increases the production of matrix metalloproteinase (MMP)-9 from C2C12 myotubes. In vivo administration of TNF-alpha in mice increased the transcript level of MMP-9 in skeletal muscle tissues. Although TNF-alpha activated all the three MAPKs (i.e. ERK1/2, JNK, and p38), inhibition of ERK1/2 or p38 but not JNK blunted the TNF-alpha-induced production of MMP-9 from myotubes. Inhibition of Akt also inhibited the TNF-alpha-induced production of MMP-9. TNF-alpha increased the activation of transcription factors NF-kappaB and AP-1 but not SP-1 in myotubes. Overexpression of a dominant negative inhibitor of NF-kappaB or AP-1 blocked the TNF-alpha-induced expression of MMP-9 in myotubes. Similarly, point mutations in AP-1- or NF-kappaB-binding sites in MMP-9 promoter inhibited the TNF-alpha-induced expression of a reporter gene. TNF-alpha increased the activity of transforming growth factor-beta-activating kinase-1 (TAK1). Furthermore, overexpression of a dominant negative mutant of TAK1 blocked the TNF-alpha-induced expression of MMP-9 and activation of NF-kappaB and AP-1. Our results also suggest that TNF-alpha induces MMP-9 expression in muscle cells through the recruitment of TRAF-2, Fas-associated protein with death domain, and TNF receptor-associated protein with death domain but not NIK or TRAF-6 proteins. We conclude that TAK1-mediated pathways are involved in TNF-alpha-induced MMP-9 production in skeletal muscle cells.

Pregnancy-associated plasma protein (PAPP)-A, a protease for IGF binding protein (IGFBP)-2, -4, and -5, may enhance IGF action by increasing its bioavailability. Here we have determined the role and mechanism of action of PAPP-A in the regulation of osteoblast proliferation in vitro and bone metabolism in vivo. Recombinant PAPP-A (100 ng/ml) significantly increased osteoblast proliferation and free IGF-I concentration. These effects were abolished by noncleavable IGFBP-4, suggesting that PAPP-A promotes osteoblast proliferation by increasing IGF bioavailability. To determine whether PAPP-A exerts an anabolic effect on bone in vivo, we developed transgenic mice that overexpress PAPP-A in osteoblasts using the 2.3-kb rat type I collagen promoter. Consistent with the increase in IGFBP-4 proteolysis, free IGF-I concentration was significantly increased in the conditioned medium of cultured osteoblasts derived from transgenic mice compared with the wild-type littermates. Calvarial bone thickness, bone marrow cavity, and skull bone mineral density were significantly increased in transgenic mice. Bone size-related parameters in femur and tibia such as total bone area and periosteal circumference as determined by peripheral quantitated computed tomography and histological analysis were significantly increased in transgenic mice. Bone formation rate and osteoid surface were increased by more than 2-fold, whereas bone resorbing surface was unaffected. These anabolic effects were sustained with aging. These findings provide strong evidence that PAPP-A acts as a potent anabolic factor in the regulation of bone formation. Thus, enhancing IGF bioavailability by PAPP-A can be a powerful strategy in the treatment of certain metabolic diseases such as osteoporosis.

Background: Pregnancy-associated plasma protein-A (PAPP-A) concentrations are increased in the circulation of patients with acute coronary syndromes (ACS) and are associated with future adverse cardiac events. PAPP-A in ACS differs from PAPP-A in pregnancy in that PAPP-A in ACS is not complexed with the proform of eosinophil major basic protein (proMBP). We investigated the effect of antibody selection on the utility of PAPP-A assays for measurement of PAPP-A in pregnancy and/or ACS, and whether immunoassays for PAPP-A in pregnancy are suitable for PAPP-A in ACS.Methods: We constructed 2-site sandwich time-resolved immunofluorometric assays using 22 monoclonal antibodies raised against pregnancy serum PAPP-A. All antibodies were studied in pairs, with each antibody used as either capture or tracer. We compared the reactivity of each antibody combination with PAPP-A/proMBP complex derived from pregnancy sera or with uncomplexed PAPP-A extracted from atherosclerotic plaques. Recombinant human PAPP-A and proMBP were also used to determine the specificity of the antibodies. We confirmed all major findings with serum samples collected from patients with myocardial infarction.Results: Six monoclonal antibodies reacted with the proMBP subunit of the PAPP-A/proMBP complex. Epitopes of 3 proMBP-reactive antibodies largely overlapped, but were well separated from those of another group of 3 proMBP-reactive antibodies. Assays using any of the 6 proMBP-reactive antibodies failed to detect PAPP-A in ACS. In addition, some 2-site assays capable of detecting PAPP-A in pregnancy were almost incapable of detecting PAPP-A in ACS, although the individual epitopes remained detectable in PAPP-A in ACS.Conclusions: Immunoassays developed for PAPP-A in pregnancy may not be suitable for PAPP-A in ACS. Assays for PAPP-A in ACS should be based on careful antibody selection and subjected to extensive testing with clinical ACS samples.

Pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, is an important regulator of mammalian growth and development. However, the role of PAPP-A and its mechanism of action in various cellular processes remain unknown. In this study, we have investigated the role of PAPP-A in skeletal myogenesis using C2C12 myoblasts. Recombinant PAPP-A was purified from the conditioned medium of HT1080 cells overexpressing PAPP-A. Treatment of C2C12 myoblasts with PAPP-A increased their proliferation in a dose- and time-dependent manner. Addition of exogenous PAPP-A also increased the myotube formation and the activity of creatine kinase in C2C12 cultures. Transient overexpression of the full-length PAPP-A-(1–1547), but not truncated protease-inactive N-terminal PAPP-A-(1–920) or C-terminal PAPP-A-(1100–1547), significantly enhanced the proliferation of C2C12 myoblasts. In vitro and in situ experiments demonstrated that PAPP-A cleaves insulin-like growth factor-binding protein (IGFBP)-2, but not IGFBP-3, in the conditioned medium of C2C12 myoblasts. Overexpression of PAPP-A led to degradation of the IGFBP-2 produced by C2C12 myoblasts and increased free IGF-I concentrations without affecting total IGF-I concentrations. Addition of protease-resistant IGFBP-4 completely abolished the PAPP-A-induced proliferation of C2C12 myoblasts. Our results demonstrate that 1) PAPP-A increases the proliferation and differentiation of myoblasts, 2) the stimulatory effect of PAPP-A on myogenesis is governed by its proteolytic activity, and 3) PAPP-A promotes skeletal myogenesis by increasing the amount of free IGFs via specific degradation of IGFBP-2 produced by myoblasts.

This study was undertaken to determine the mechanism by which proform of eosinophil major basic protein (proMBP) inhibits the IGFBP-4 proteolytic activity of pregnancy-associated plasma protein (PAPP)-A. Co-overexpression of PAPP-A with proMBP in 293T cells, or co-incubation of 293T cells, respectively, overexpressing proMBP and PAPP-A resulted in the formation of a covalent proMBP–PAPP-A complex and inhibition of IGFBP-4 proteolysis. Similar results were obtained when recombinant proMBP and PAPP-A were incubated in the presence of U2 osteosarcoma cells or when recombinant proMBP was added to the U2 cells overexpressing PAPP-A. In contrast, no formation of covalent proMBP–PAPP-A complex or inhibition of IGFBP-4 proteolysis was observed when recombinant proMBP and PAPP-A were incubated under cell-free conditions, although proMBP was able to interact with PAPP-A in a non-covalent manner. These new findings suggest that formation of covalent proMBP–PAPP-A complex is a cell-mediated event and is required for proMBP to inhibit the catalytic activity of PAPP-A.

We have previously obtained compelling proof-of-principle evidence for COX2 gene therapy for fracture repair using integrating retroviral vectors. For this therapy to be suitable for patient uses, a suitable vector with high safety profile must be used. Accordingly, this study sought to evaluate the feasibility of AAV as the vector for this COX2 gene therapy, because AAV raises less safety issues than the retroviral vectors used previously. However, an appropriate AAV serotype is required to provide early increase in and adequate level of COX2 expression that is needed for fracture repair. Herein, we reported that AAV-DJ, an artificial AAV pseudoserotype, is highly effective in delivering COX2 gene to fracture sites in a mouse femoral fracture model. Compared with AAV-2, the use of AAV-DJ led to ~5-fold increase in infectivity in mesenchymal stem cells (MSCs) and provided an earlier and significantly higher level of transgene expression at the fracture site. Injection of this vector at a dose of 7.5 × 10(11) genomic copies led to high COX2 level at the fracture site on day 3 after injections and significantly promoted fracture union at 21 days, as analyzed by radiography and μ-CT. The therapeutic effect appears to involve enhanced osteoblastic differentiation of MSCs and remodeling of callus tissues to laminar bone. This interpretation is supported by the enhanced expression of several key genes participating in the fracture repair process. In conclusion, AAV-DJ is a promising serotype for the AAV-based COX2 gene therapy of fracture repair in humans.

Pregnancy-associated plasma protein-A (PAPP-A) has been identified as the insulin-like growth factor (IGF)-dependent IGF-binding protein-4 (IGFBP-4) protease produced by human fibroblasts. Recently, we found that serum proteases induced during human pregnancy cleaved IGFBP-4 in both an IGF-II-dependent and an IGF-II-independent fashion. This study sought to determine whether PAPP-A is the predominant IGFBP-4 protease in human pregnancy serum (PS) and to assess the in vitro role of serum PAPP-A. Immunoprecipitation with PAPP-A antibody effectively depleted PAPP-A from the PS and completely abolished both IGF-II-dependent and IGF-II-independent IGFBP-4 proteolytic activity in PS. Direct addition of PAPP-A antibody to PS completely blocked IGFBP-4 proteolysis and partially blocked IGFBP-5 proteolysis, but had no effect on IGFBP-3 proteolysis. To evaluate the role of serum PAPP-A, we tested whether PAPP-A in PS modulated the inhibitory activity of IGFBP-4 on IGF-II-induced cell proliferation in human osteosarcoma MG63 cells. The wild-type IGFBP-4 (WTBP-4; 200 ng/mL) failed to inhibit proliferation of the cells treated with PS (0.1% or 0.3%) alone or in combination with IGF-II (40 ng/mL), whereas the inhibitory effect of WTBP-4 was observed in the cells treated with nonpregnancy serum alone or in combination with IGF-II (P < 0.05). In contrast to WTBP-4, a protease-resistant IGFBP-4 was able to inhibit proliferation of the cells treated with PS alone or in combination with IGF-II (P < 0.05). In the presence of PAPP-A neutralizing antibody, the inhibitory effect of WTBP-4 on proliferation of the cells treated with IGF-II and PS was restored. In summary, these data demonstrate 1) that PAPP-A represents the predominant IGFBP-4 protease in PS; 2) that PAPP-A may in part contribute to IGFBP-5, but not IGFBP-3, proteolytic activity in PS; and 3) that PAPP-A enhances the bioactivity of IGFs in vitro by degrading IGFBP-4.

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4), a consistent inhibitor of IGF action, is subject to proteolytic cleavage by the IGF-II-dependent IGFBP-4 protease. However, regulation of the IGF-II-dependent IGFBP-4 protease in vivo is not known. As IGFBP proteases are known to be triggered during pregnancy, we systematically evaluated the changes in IGFBP-4 proteolysis by serum collected throughout human pregnancy. Results from in vitro protease assays using recombinant IGFBP-4 revealed that IGFBP-4 proteolysis determined in both the presence and absence of exogenous IGF-II significantly increased during the first and second trimesters and reached a plateau by the third trimester. However, in the absence of IGF-II, IGFBP-4 proteolysis by pregnancy serum was only observed after prolonged incubation. IGF-II dose dependently increased IGFBP-4 proteolysis by pregnancy serum, with maximal stimulation observed at a concentration of 0.7 mol/L relative to IGFBP-4. In contrast, IGF-II at an equimolar...

To characterize the insulin-like growth factor binding protein-4 (IGFBP-4) protease produced by human osteoblasts (hOBs), we localized and determined the role of the proteolytic domains in human IGFBP-4 (hIGFBP-4) in modulating IGF-II actions. N-terminal amino acid sequence and mass spectrometric analyses of the 6xHis-tagged IGFBP-4 proteolytic fragments revealed that Met135-Lys136 was the only cleavage site recognized by the IGF-II-dependent IGFBP-4 protease produced by hOBs. This cleavage site was confirmed by the finding that deletion of His121 to Pro141 blocked proteolysis. However, unexpectedly, deletion of Pro94 to Gln119 containing no cleavage site had no effect on IGF-II binding activity but blocked proteolysis. Addition of the synthetic peptide corresponding to this region at concentrations of 250 or 1000 molar excess failed to block IGFBP-4 proteolysis. These data suggest that residues 94-119 may be involved in maintaining the IGFBP-4 conformation required to expose the cleavage site rather than being involved in direct protease-substrate binding. To determine the physiological significance of the IGF-II-dependent IGFBP-4 protease, we compared the effect of the wild-type IGFBP-4 and the protease-resistant IGFBP-4 analogs in blocking IGF-II-induced cell proliferation in normal hOBs, which produce IGFBP-4 protease, and MG63 cells, which do not produce IGFBP-4 protease. It was found that protease-resistant IGFBP-4 analogs were more potent than the wild-type protein in inhibiting IGF-II-induced cell proliferation in hOBs but not in MG63 cells. These data suggest that IGFBP-4 proteolytic fragments are not biologically active and that IGFBP-4 protease plays an important role in regulating IGFBP-4 bioavailability and consequently the mitogenic activity of IGFs in hOBs.

More than 3 kb of the human (h)IGFBP-4 gene 5′-flanking region was sequenced and assessed for promoter activity. The hIGFBP-4 promoter resides within a CpG island and demonstrates strong basal activity in human osteoblast-like osteosarcoma and COS-7 monkey kidney cells. Transient transfection of cells with hIGFBP-4 promoter-linked deletion constructs demonstrated that multiple cooperating cis-acting elements within 836 bp of the 5′-flanking region contributed to overall promoter strength.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) suppresses autoimmunity and inflammation; however, the mechanism of its action has not been fully understood. We sought in this study to determine whether the anti-immune/anti-inflammatory action of 1,25(OH)2D3 is in part mediated through an interplay between 1,25(OH)2D3 and toll-like receptor (TLR)7/8 signaling. 1,25(OH)2D3 treatment prior to and/or following experimental autoimmune encephalomyelitis (EAE) induction effectively reduced inflammatory cytokine expression in the spinal cord and ameliorated EAE. These effects were accompanied with a reduction in expression of several TLRs with the most profound effect observed for TLR8. The expression of TLR8 adaptor protein MyD88 was also significantly reduced by 1,25(OH)2D3. To determine the molecular mechanism by which 1,25(OH)2D3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we evaluated whether 1,25(OH)2D3 can directly inhibit TLR8 signaling and the resulting inflammatory responses in human THP-1 monocytes. 1,25(OH)2D3 treatment not only significantly reduced TLR8 expression but also the expression or activity of MyD88, IRF-4, IRF-7 and NF-kB in monocytes challenged with TLR8 ligands. TLR8 promoter-luciferase reporter assays indicated that 1,25(OH)2D3 decreases TLR8 mRNA level in part via inhibiting TLR8 gene transcription activity. As a result of inhibition on TLR8 signaling cascade at various stages, 1,25(OH)2D3 significantly diminished the TLR8 target gene expression (TNF-α and IL-1β). In summary, our novel findings suggest that TLR8 is a new target of 1,25(OH)2D3 and may mediate the anti-inflammatory action of 1,25(OH)2D3. Our findings also point to a destructive role of TLR8 in EAE and shed lights on pathogenesis of multiple sclerosis.

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.

While there is compelling evidence for an anabolic role of PAPP-A, an IGFBP protease, in muscle development, its effect on dynamic regulation of muscle regeneration has not been investigated. In this study, we evaluated the effect of transgenic PAPP-A overexpression in skeletal muscle of mice on myofiber formation in intact and crush-injured tibialus anterior muscle.Skeletal muscle in transgenic mice overexpressing human PAPP-A in skeletal muscle was subjected to crush-injury. Myofiber formation and myogenic gene expression were then evaluated in injured or intact muscle of PAPP-A transgenic mice and wild-type mice.In the intact muscle, aging PAPP-A transgenic (Tg.) mice (age of 12 months) showed more than a 2-fold increase in both myofiber size and number of nuclei per myofiber compared with their wild-type (Wt.) littermates. Myofibers with centered nuclei, a hallmark of muscle regeneration, were increased from1% in Wt. mice to 65% in Tg. muscle. In the injured muscle, reduced inflammatory cell infiltration and increased new myofiber size and the area occupied by new myofibers were observed in PAPP-A transgenic mice compared to wild-type littermates. MyoD and creatine kinase in the injured muscle was also significantly increased in the Tg. mice. Although TNF-α induced PAPP-A expression in skeletal myoblast culture and its expression increased upon injury, abrogation of TNF-α signaling in TNF-α receptor knockout mice had no impact on the extent of injury induction of PAPP-A. We also found that TGF-β expression was significantly increased following muscle injury in vivo and treatment with recombinant TGF-β in vitro significantly enhanced PAPP-A expression in skeletal myoblasts.Our findings demonstrate that exogenous PAPP-A can promote recovery of muscle injury in aging mice albeit the expression of endogenous PAPP-A had already been increased dramatically upon muscle injury.

Although IGFs are indispensable to skeletal muscle development, little information is available regarding the mechanisms regulating the local action of IGFs in skeletal muscle tissues. Here we tested the hypothesis that pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, promotes skeletal muscle formation in vivo through degrading IGF binding proteins (IGFBPs), which increases the bioavailability of IGFs. Expression of PAPP-A is significantly increased in muscle five days after muscle injury in mice. Targeted overexpression of PAPP-A using a muscle-specific promoter significantly increased the prenatal/postnatal growth, skeletal muscle weight, and muscle fiber area in mice. These anabolic effects were reproduced using F2/F3 progeny. Free IGF-I concentration was severalfold higher in the conditioned medium (CM) of ex vivo cultured muscle from the transgenic mice, compared with the wild-type littermate muscle. Accordingly, the proliferation of C2C12 myoblasts was significantly increased in the presence of CM from cultured skeletal muscle of the transgenic mice, compared with the controls. This observed increase in myoblast proliferation was abolished on addition of noncleavable IGFBP-4 peptide, which reduced free IGF-I concentration back to the basal level of the wild-type CM. Furthermore, proliferation and differentiation of C2C12 myoblasts was increased by transient overexpression of proteolytically active PAPP-A but not by inactive mutant PAPP-A (E483/A). Collectively, we identified PAPP-A as a novel regulator of prenatal/postnatal growth and skeletal muscle formation in vivo. Moreover, our studies provide the first experimental evidence that IGFBP degradation is a key determinant in modulating the local action of IGFs in muscle.

TWEAK cytokine has been implicated in several biological responses including inflammation, angiogenesis, and osteoclastogenesis. We have investigated the role of TWEAK in regulating skeletal muscle mass. Addition of soluble TWEAK protein to cultured myotubes reduced the mean myotube diameter and enhanced the degradation of specific muscle proteins such as CK and MyHCf. The effect of TWEAK on degradation of MyHCf was stronger than its structural homologue, TNF-alpha. TWEAK increased the ubiquitination of MyHCf and the transcript levels of atrogin-1 and MuRF1 ubiquitin ligases. TWEAK inhibited phosphorylation of Akt kinase and its downstream targets GSK-3beta, FOXO1, mTOR, and p70S6K. Furthermore, TWEAK increased the activation of NF-kappaB transcription factor in myotubes. Adenoviral-mediated overexpression of IkappaB alpha deltaN (a degradation-resistant mutant of NF-kappaB inhibitory protein IkappaB alpha) in myotubes blocked the TWEAK-induced degradation of MyHCf. Chronic administration of TWEAK in mice resulted in reduced body and skeletal muscle weight with an associated increase in the activity of ubiquitin-proteasome system and NF-kappaB. Finally, muscle-specific transgenic overexpression of TWEAK decreased the body and skeletal muscle weight in mice. Collectively, our data suggest that TWEAK induces skeletal muscle atrophy through inhibition of the PI3K/Akt signaling pathway and activation of the ubiquitin-proteasome and NF-kappaB systems.

PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP-PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a approximately 470 kDa PAPP-A form (PAPP-A470) to a approximately 400 kDa PAPP-A form (PAPP-A400). Since unreduced PAPP-A400 co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A470 and PAPP-A400 co-migrated on SDS/PAGE, conversion of PAPP-A470 to PAPP-A400 is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A400/PAPP-A470 is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A400 exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A470. These data suggest that a novel mechanism, namely conversion of PAPP-A470 to the less-active PAPP-A400, could account for the TPA-induced suppression of PAPP-A activity.

Serum IGFBP-4 proteolytic activity increases dramatically during human pregnancy and is mainly attributed to the pregnancy-associated plasma protein-A (PAPP-A). To understand the regulation and actions of PAPP-A in vivo, we evaluated the utility of a mouse model system. Serum from day-9 and day-17 pregnant mice and age-matched controls was tested for IGFBP-4 proteolytic activity using recombinant mouse IGFBP-4 as the substrate. Surprisingly, IGFBP-4 proteolytic activity in mouse pregnancy serum (mPS) was not significantly different from that of non-pregnancy serum (mNPS). Addition of IGF-II to mPS or mNPS at a dose sufficient to increase IGFBP-4 proteolysis by human PS failed to enhance IGFBP-4 proteolysis. PAPP-A neutralization antibody did not inhibit IGFBP-4 proteolysis by mPS or mNPS, but completely blocked IGFBP-4 proteolytic activity in human PS (hPS, mouse osteoblast conditioned medium, and mouse amniotic fluid). To determine whether the lack of PAPP-A activity in mPS was due to low expression of PAPP-A in the placenta, we cloned a mouse genomic DNA, which contained 1 kb of the entire exon 2 coding sequence and 2.5 kb of the flanking intron sequences. The exon 2-coded mouse and human PAPP-A shared 86% amino acid sequence identity. RT-PCR analysis revealed that the PAPP-A mRNA level in mouse placenta was lower compared to that in human placenta by at least two orders of magnitude. PAPP-A expression was also lower in mouse placenta compared to those in mouse kidney, osteoblasts, and bone marrow stromal cells. Conclusions: (1) Serum IGFBP-4 proteolytic activity is differentially regulated by pregnancy in human and mouse. (2) The lack of an increase in serum IGFBP-4 proteolytic activity during mouse pregnancy is due to the low level of PAPP-A expression in the placenta.

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is a potent inhibitor of IGF actions in vitro. However, we found that systemic administration of IGFBP-4 at pharmacological doses caused a significant increase in bone formation parameters in mice by a mechanism that may involve increased IGF bioavailability via proteolysis of IGFBP-4. To evaluate the hypothesis that proteolysis of IGFBP-4 is essential for the stimulatory effects of systemically administered IGFBP-4, we produced wild-type, protease-resistant, and IGFBP-4 proteolytic fragments and evaluated their effects using biochemical markers. Protease-resistant IGFBP-4 was more potent than wild-type IGFBP-4 in inhibiting IGF-I-induced mouse osteoblast cell proliferation in vitro and in inhibiting IGF-I-induced increase in alkaline phosphatase (ALP) activity in bone extract after local administration in vivo. Systemic administration of wild-type IGFBP-4, but not protease-resistant IGFBP-4, increased serum osteocalcin, serum ALP, and ALP in skeletal extracts in a dose-dependent manner, with a maximal effect of 40% (P < 0.05) at 1.25 nmol/mouse. Systemic administration of wild-type, but not protease-resistant, IGFBP-4 increased free IGF-I levels in serum in normal mice. IGF-I, but not wild-type IGFBP-4, increased bone formation parameters in IGF-I-deficient mice. This study demonstrates that systemic administration of IGFBP-4 increases bone formation parameters in mice by increasing IGF bioavailability in the circulation via an IGFBP-4 protease-dependent mechanism.

Our previous findings suggest that binding of IGF binding protein-4 (IGFBP-4) to IGFs is essential for the inhibitory effect of IGFBP-4 on the activity of IGFs, both in vitro and in vivo. Therefore, understanding the structural determinants of IGF binding in IGFBP-4 is important to the general understanding of the biology of the IGF system. This study sought to further localize the IGF binding domain and to evaluate the role of Cys residues in IGF binding. Our data revealed that full-length IGFBP-4 peptides lacking the residues Leu(72)-Ser(91) or Leu(72)-His(74) or Gly(75)-Ser(91) failed to bind to IGF-I or IGF-II, whereas deletion of the residue Leu(72) or residues Met(80)-Ser(91) led to a 2- to 3-fold reduction in IGF-I and IGF-II binding activity. The IGF-I and IGF-II binding activities were dramatically reduced by the single mutation, Cys9/Arg (>25-fold), and to a lesser degree, by the single mutation, Cys12/Arg (the first N-terminal Cys residue was designated Cys1). The mutation Cys17/Ser or Cys18/Tyr or Cys20/Ser each resulted in a similar but moderate ( approximately 5-fold) reduction in IGF-II binding activity. The IGF-I binding activity was also dramatically reduced by the mutation Cys18/Tyr, and to a lesser extent, by the mutation Cys17/Ser or Cys20/Ser. These data suggest: 1) the IGF-I and IGF-II binding domain in IGFBP-4 involves a hydrophobic motif (Leu(72)-Met(80)) located in the distal part of the conserved N-terminal region, and 2) the N-terminal Cys residues (Cys9 and Cys12) are more critical than the C-terminal Cys residues (Cys17 and Cys20) in affecting the IGF-I and IGF-II binding. Based on these data, we speculate that the structural determinants of IGF-I and IGF-II binding in IGFBP-4 are very similar, if not identical.

Bone volume is determined by the relative rates of bone formation and bone resorption. Recent research in several laboratories suggests that growth factors may act locally to modulate bone formation by stimulating osteoblast proliferation and activity. A number of bone-derived growth factors have been isolated and characterized from bone matrix extracts and from media conditioned by bone cells and bone organs in culture. The growth factors found in bone matrix include insulinlike growth factors I and II, transforming growth factor-beta, acidic and basic fibroblast growth factor, platelet-derived growth factor, and bone morphogenetic proteins. Conditioned medium from bone cells contains several of these growth factors and also hematopoietic factors. These bone matrix-derived growth factors have different biologic activities, including mitogenic, differentiating, chemotactic, and osteolytic activities. Evidence suggests that bone cells produce substantial quantities of growth factors for extracellular storage in bone matrix. Apart from being produced for extracellular storage, it is possible that growth factors secreted by bone cells have acute effects on their neighboring osteoblastic cells, i.e., paracrine action, or on themselves, i.e., autocrine action. The release of matrix-stored growth factors by bone resorption may mean that growth factors act as delayed paracrine agents, e.g., osteoblasts deposit growth factors in bone and later when these growth factors are released from bone via bone resorption, the growth factors stimulate osteoblast precursors to proliferate. The findings that bone is a storehouse for growth factors and that bone cells in culture produce and respond to bone growth factors suggest bone growth factors may act as potential determinants of local bone formation. This review is focused on the structure, regulation, and biologic actions of the known bone growth factors.

To identify the molecular mechanism by which insulin-like growth factor binding protein-4 (IGFBP-4) exerts its inhibitory effects on insulin-like growth factor (IGF) actions, we localized and determined the role of the IGF binding domain in modulating IGF actions in human osteoblasts. Deletion analysis using IGFBP-4 expressed in bacteria revealed that the N-terminal sequence Leu72-Ser91 was essential for IGF binding. The C-terminal fragments (His121-Glu237 or Arg142-Glu237) did not bind to IGF but loss of these regions decreased IGF binding activity. Detailed deletion analysis identified the residues Cys205-Val214 as the motif to facilitate IGF binding. Mitogenic studies revealed that an IGFBP-4 mutant (His74 replaced by Pro74) and an N-terminal peptide (N terminus to Thr71) with little IGF binding activity failed to inhibit IGF-II-induced human osteoblast proliferation. An N-terminal peptide (N terminus to Asn182) with reduced IGF binding activity inhibited IGF action but with lower potency. In contrast, an IGFBP-4 mutant (His74 replaced with Ala74) exhibited similar IGF binding activity and potency in inhibiting the activity of IGF-II compared with the wild type. Therefore, the N-terminal sequence (Leu72-Ser91) and the C-terminal sequence (Cys205-Val214) are necessary to form the high affinity IGF binding domain, which is the major structural determinant of the IGFBP-4 function.

To test if ephrin B1 overexpression enhances bone mass, we generated transgenic mice overexpressing ephrin B1 under the control of a 3.6 kb rat collagen 1A1 promoter (Col3.6-Tg (efnb1) ). Col3.6-Tg (efnb1) mice express 6-, 12- and 14-fold greater levels of full-length ephrin B1 protein in bone marrow stromal cells, calvarial osteoblasts, and osteoclasts, respectively. The long bones of both genders of Col3.6-Tg (efnb1) mice have increased trabecular bone volume, trabecular number, and trabecular thickness and decreased trabecular separation. Enhanced bone formation and decreased bone resorption contributed to this increase in trabecular bone mass in Col3.6-Tg (efnb1) mice. Consistent with these findings, our in vitro studies showed that overexpression of ephrin B1 increased osteoblast differentiation and mineralization, osterix and collagen 1A1 expression in bone marrow stromal cells. Interaction of ephrin B1 with soluble clustered EphB2-Fc decreased osteoclast precursor differentiation into multinucleated cells. Furthermore, we demonstrated that the mechanical loading-induced increase in EphB2 expression and newly formed bone were significantly greater in the Col3.6-Tg (efnb1) mice than in WT littermate controls. Our findings that overexpression of ephrin B1 in bone cells enhances bone mass and promotes a skeletal anabolic response to mechanical loading suggest that manipulation of ephrin B1 actions in bone may provide a means to sensitize the skeleton to mechanical strain to stimulate new bone formation.

This study tested the hypothesis that 1,25-dihydroxyvitamin D3 treatment alters the pattern of protein phosphorylation in rat kidney. Treatment with 1,25-dihydroxyvitamin D3 (100 ng/day s.c. x 7d) in vitamin D-deficient rats markedly decreased phosphorylation of a particulate protein (91 +/- 0.6 kDa, n = 9) and moderately increased phosphorylation of a cytosolic protein (108 +/- 0.8 kDa, n = 9) in the kidney. The decreased phosphorylation of the 91-kDa particulate protein showed a graded dose response (0-200 ng/day), as did the more moderate increase in phosphorylation of the 108-kDa cytosolic protein. In conclusion, this study has provided evidence that 1,25-dihydroxyvitamin D3 plays an important role in the regulation of protein phosphorylation in rat kidney and adds these biochemical events to the growing list of 1,25-dihydroxyvitamin D3 effects in this poorly understood target tissue.