ABSTRACT
Transforming growth factor-beta (TGF-beta) is known to regulate chondrocyte proliferation and hypertrophic differentiation in embryonic bone cultures by a perichondrium dependent mechanism. To begin to determine which factors in the perichondrium mediate the effects of TGF-beta, we studied the effect of Insulin-like Growth Factor-1 (IGF-I) and Fibroblast Growth Factors-2 and -18 (FGF2, FGF18) on metatarsal organ cultures. An increase in chondrocyte proliferation and hypertrophic differentiation was observed after treatment with IGF-I. A similar effect was seen after the perichondrium was stripped from the metatarsals suggesting IGF-I acts directly on the chondrocytes. Treatment with FGF-2 or FGF-18 resulted in a decrease in bone elongation as well as hypertrophic differentiation. Treatment also resulted in a decrease in BrdU incorporation into chondrocytes and an increase in BrdU incorporation in perichondrial cells, similar to what is seen after treatment with TGF-beta1. A similar effect was seen with FGF2 after the perichondrium was stripped suggesting that, unlike TGF-beta, FGF2 acts directly on chondrocytes to regulate proliferation and hypertrophic differentiation. To test the hypothesis that TGF-beta regulates IGF or FGF signaling, activation of the receptors was characterized after treatment with TGF-beta. Activation was measured as the level of tyrosine phosphorylation on the receptor. Treatment with TGF-beta for 24h did not alter the level of IGFR-I tyrosine phosphorylation. In contrast, treatment with TGF-beta resulted in and increase in tyrosine phosphorylation on FGFR3 without alterations in total FGFR3 levels. TGF-beta also stimulated expression of FGF18 mRNA in the cultures and the effects of TGF-beta on metatarsal development were blocked or partially blocked by pretreatment with FGF signaling inhibitors. The results suggest a model in which FGF through FGFR3 mediates some of the effects of TGF-beta on embryonic bone formation.
Subject(s)
Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factors/pharmacology , Metatarsal Bones/cytology , Metatarsal Bones/drug effects , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacology , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Chondrocytes/cytology , Chondrocytes/drug effects , Fibroblast Growth Factor 2/antagonists & inhibitors , Fibroblast Growth Factors/antagonists & inhibitors , Gene Expression Regulation, Developmental/drug effects , Hindlimb/cytology , Hindlimb/embryology , Insulin-Like Growth Factor I/pharmacology , Metatarsal Bones/embryology , Metatarsal Bones/metabolism , Mice , Organ Culture Techniques , Receptors, Fibroblast Growth Factor/metabolismABSTRACT
T cell depletion plus donor bone marrow cell (BMC) infusion induces long-term kidney allograft survival in a limited number of rhesus macaque recipients. Therefore, there is a need to enhance the tolerogenic activity of donor BMCs. The tolerogenic effect of donor BMCs is ascribed to a veto activity, mediated by a CD8+ subset that upregulates immunoregulatory effector molecules, transforming growth factor-beta1 (TGF-beta), and FasL, after interaction with donor-reactive cytotoxic T lymphocyte precursors (CTLp), leading to clonal inactivation/deletion of donor-reactive CTLp. Of note, the receptors for TGF-beta1- and FasL-induced signal transduction are upregulated in activated T cells. Since mature dendritic cells (DCs) are exceptionally efficient activators of T cells, we postulated that mature DCs modified to overexpress TGF-beta1 and FasL might exert potent veto (i.e., inactivating/deleting) activity independent of CD8 expression. A fusion protein comprising antihuman CD40 single-chain antibody and soluble coxsackie-adenovirus receptor enabled high-efficiency transduction of rhesus monocyte-derived DCs (Rh MDDCs) by recombinant adenovirus (Ad). Mature Rh MDDCs transduced with Ad encoding active TGF-beta1 retained a mature phenotype yet exhibited potent alloantigen-specific cellular immunosuppression. Such modified MDDCs have the potential to promote tolerance induction to allografts in vivo.
