Exact coordination of growth plate chondrocyte proliferation is necessary for normal endochondral bone development and growth. Here we show that PTHrP and TGF␤ control chondrocyte cell cycle progression and proliferation by stimulating signaling pathways that activate transcription from the cyclin D1 promoter. The TGF␤ pathway activates the transcription factor ATF-2, whereas PTHrP uses the related transcription factor CREB, to stimulate cyclin D1 promoter activity via the CRE promoter element. Inhibition of cyclin D1 expression with antisense oligonucleotides causes a delay in progression of chondrocytes through the G1 phase of the cell cycle, reduced E2F activity, and decreased proliferation. Growth plates from cyclin D1-deficient mice display a smaller zone of proliferating chondrocytes, confirming the requirement for cyclin D1 in chondrocyte proliferation in vivo. These data identify the cyclin D1 gene as an essential component of chondrocyte proliferation as well as a fundamental target gene of TGF␤ and PTHrP during skeletal growth. INTRODUCTIONEndochondral bone growth is controlled by the coordinated proliferation and differentiation of growth plate chondrocytes (Cancedda et al., 1995). Numerous skeletal diseases (chondrodysplasias) are caused by genetic disturbances of these processes, resulting in skeletal deformities, dwarfism, and early onset osteoarthritis Olsen, 1997a, 1997b). Despite the recent identification of many genes that control chondrocyte proliferation and differentiation Olsen, 1997a, 1997b;Beier et al., 1999a), the intracellular signaling pathways and transcriptional mechanisms involved are not well defined. Transforming growth factor beta (TGF␤) and parathyroid hormone-related peptide (PTHrP) stimulate the proliferation of chondrocytes and chondrosarcoma cells in vitro (O'Keefe et al., 1988;Rosier et al., 1989;Guerne and Lotz, 1991;Loveys et al., 1993;Guerne et al., 1994). In addition, interruption of TGF␤ or PTHrP signaling in vivo in mice causes a reduction in the number of proliferative chondrocytes as well as premature differentiation of these cells (Amizuka et al., 1994;Karaplis et al., 1994;Serra et al., 1997;Yang et al., 2001). These data suggest that both factors are required for normal chondrocyte proliferation in vivo and in vitro. However, the intracellular signaling pathways activated by PTHrP and TGF␤ in chondrocytes as well as their target genes have yet to be identified.Cell cycle genes appear to play an important role in the control of chondrocyte proliferation and differentiation (Beier et al., 1999a;LuValle and Beier, 2000). Progression through the eukaryotic cell cycle is controlled by the activity of a family of kinases called the cyclin-dependent kinases or CDKs (Weinberg, 1995). CDK activity is strictly controlled by a number of mechanisms, including phosphorylation status and the presence of inhibitory proteins such as p16Ink4 or p21 Cip1/Waf1 . An absolute requirement for the activity of a given CDK is the presence of its specific cyclin partner. The D-type c...

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Our results demonstrate that CD4(+) T cells, B cells and macrophages are the immune cells recruited to and involved in the rejection of encapsulated NPI. Immune molecules secreted by these cells as well as complement can traverse the microcapsule membrane and are responsible for destroying the NPI cells. Treatment regimens which target these molecules may modify the rejection of encapsulated NPI and lead to prolonged islet xenograft survival.

We previously demonstrated that short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) induces tolerance to neonatal porcine islet (NPI) xenografts that is mediated by regulatory T cells (Tregs) in B6 mice. In this study, we examined whether the coinhibitory molecule PD-1 is required for the induction and maintenance of tolerance to NPI xenografts. We also determined whether tolerance to NPI xenografts could be extended to allogeneic mouse or xenogeneic rat islet grafts since we previously demonstrated that tolerance to NPI xenografts could be extended to second-party NPI xenografts. Finally, we determined whether tolerance to NPI xenografts could be extended to allogeneic mouse or second-party porcine skin grafts. Diabetic B6 mice were transplanted with 2,000 NPIs under the kidney capsule and treated with short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs. Some of these mice were also treated simultaneously with anti-PD-1 mAb at >150 days posttransplantation. Spleen cells from some of the tolerant B6 mice were used for proliferation assays or were injected into B6 rag −/− mice with established islet grafts from allogeneic or xenogeneic donors. All B6 mice treated with anti-LFA-1 and anti-CD154 mAbs achieved and maintained normoglycemia until the end of the study; however, some mice that were treated with anti-PD-1 mAb became diabetic. All B6 rag −/− mouse recipients of first-and second-party NPIs maintained normoglycemia after reconstitution with spleen cells from tolerant B6 mice, while all B6 rag −/− mouse recipients of allogeneic mouse or xenogeneic rat islets rejected their grafts after cell reconstitution. Tolerant B6 mice rejected their allogeneic mouse or xenogeneic second-party porcine skin grafts while remaining normoglycemic until the end of the study. These results show that porcine islet-specific tolerance is dependent on PD-1, which could not be extended to skin grafts.

To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-alpha and IFN-gamma or stimulation with LPS demonstrated a time- and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and +128 bp was found to mediate IFN-gamma, TNF-alpha, and LPS responsiveness as well as the synergistic effect of IFN-gamma/TNF-alpha on hBf promoter activity. Site-directed mutagenesis of a IFN-gamma-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-gamma responsiveness. Mutagenesis of a nuclear factor (NF)-kappaB cis element at -466 to -456 bp abrogated TNF-alpha and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-gamma, TNF-alpha, and LPS is through GAS and NF-kappaB cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway.