Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.8 cells, in which both genes are expressed, were transfected with reporter constructs driven by native (i.e. wild-type) rat OC and mouse OP promoters. TGF beta abrogated the 1,25-(OH)2D3 enhanced transcription of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions from proximal basal regulatory elements. These transcriptional effects were further investigated for contribution of the trans-activating factors, which interact with OC and OP VDREs, involving the vitamin D receptor (VDR) and retinoid X receptor (RXR). Gel mobility shift assays show that TGF beta significantly reduces induction of the heterodimers VDR/RXR complexes in 1,25-(OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analysis reveal that TGF beta does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGF beta on basal transcription. Our studies demonstrate that the inhibitory action of TGF beta on the 1,25-(OH)2D3 enhancement of OC and OP transcription in osteoblastic cells results from modulations of protein-DNA interactions at the OC and OP VDRE, which cannot be accounted for by changes in VDR protein levels. As OC and OP participate in bone turnover, our results provide insight into the contributions of TGF beta and 1,25-(OH)2D3 to VDR-mediated gene regulatory mechanism operative in bone formation and/or resorption events.
The Australian skink Egernia stokesii is parasitized uncommonly by Plasmodium circularis n. sp. and by Plasmodium mackerrasae. Plasmodium circularis is distinguished from all other plasmodiids by immature schizonts that encircle host cell nuclei, forming an unbroken ring from apparent fusion of the attenuated ends. Mature schizonts contract into halteridial or dumbbell-shaped forms 15.6 x 4.3 microm, LW 66.2 microm2, with 19-52 nuclei. Rounded or oval gametocytes are 9.0 x 7.3 microm, LW 66.9 microm2, and L/W 1.24. Gametocyte LW is 2.63 x host erythrocyte nucleus size and 1.79X uninfected erythrocyte nuclei. Plasmodium mackerrasae occurs in high prevalence and often massive parasitemia in E. stokesii. Schizonts, often oblong, elongate, or oval, are 5.1 x 3.7 microm, LW 19.8 microm2, with 7.2 merozoites. Immature gametocytes, elongate with terminal nucleus, may produce multiple infections of 6 or more parasites. Mature gametocytes, usually rounded, are 5.8 x 4.6 microm, LW 26.7 microm2, and L/W 1.29. Gametocyte size is 0.98 x host erythrocyte nucleus size and 1.03 x uninfected erythrocyte nuclei. Phanerozoites, in endothelium or connective tissue of most organs, may appear in large numbers in circulating blood as seemingly intact bodies of regular form, similar to or larger than phanerozoites seen in sections. Previously unreported phenomena for hemosporidian parasites include extremely large, highly irregular exoerythrocytic schizonts, in circulating blood, perhaps torn from endothelial lining of blood vessels and sinuses, and a visible flooding of free merozoites into the blood stream.
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