Bone quality is an important determinant of osteoporosis, and proper osteoblast differentiation plays an important role in the control and maintenance of bone quality. We investigated the impact of activin signaling on human osteoblast differentiation, extracellular matrix formation, and mineralization. Activins belong to the transforming growth factor-beta superfamily and activin A treatment strongly inhibited mineralization in osteoblast cultures, whereas the activin antagonist follistatin increased mineralization. Osteoblasts produced activin A and follistatin in a differentiation-dependent manner, leading to autocrine regulation of extracellular matrix formation and mineralization. In addition, mineralization in a vascular smooth muscle cell-based model for pathological calcification was inhibited. Comparative activin A and follistatin gene expression profiling showed that activin signaling changes the expression of a specific range of extracellular matrix proteins prior to the onset of mineralization, leading to a matrix composition with reduced or no mineralizing capacity. These findings demonstrate the regulation of osteoblast differentiation and matrix mineralization by the activin A-follistatin system, providing the possibility to control bone quality as well as pathological calcifications such as atherosclerosis by using activin A, follistatin, or analogs thereof.
Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1Alpha-hydroxylation of 25-(OH)D3 to form the bioactive vitamin D hormone, 1alpha,25-(OH)2D3, is classically considered to take place in the kidney. However, 1alpha-hydroxylase has been reported at extrarenal sites. Whether bone is a 1alpha,25-(OH)2D3 synthesizing tissue is not univocal. The aim of this study was to investigate an autocrine/paracrine function for 1alpha,25-(OH)2D3 in bone. We show that 1alpha-hydroxylase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1alpha-hydroxylase substrate 25-(OH)D3, the osteoblasts can produce sufficient 1alpha,25-(OH)2D3 to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1alpha-hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1alpha-hydroxylase in osteoblasts. In contrast, interleukin (IL)-1beta strongly induces 1alpha-hydroxylase. Besides the bone-forming cells, we demonstrate 1alpha-hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1alpha-hydroxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone.
Vitamin D plays a major role in the regulation of mineral homeostasis and affects bone metabolism. So far, detailed knowledge on the vitamin D endocrine system in human bone cells is limited. Here we investigated the direct effects of 1alpha,25-(OH)2D3 on osteoblast differentiation and mineralization. Also, we studied the impact of 24-hydroxylation, generally considered as the first step in the degradation pathway of vitamin D, as well as the role of the nuclear and presumed membrane vitamin D receptor (VDR). For this we used a human osteoblast cell line (SV-HFO) that has the potency to differentiate during culture forming a mineralized extracellular matrix in a 3-week period. Transcriptional analyses demonstrated that both 1alpha,25-(OH)2D3 and the 24-hydroxylated metabolites 24R,25-(OH)2D3 and 1alpha,24R,25-(OH)3D3 induced gene transcription. All metabolites dose-dependently increased alkaline phosphatase (ALP) activity and osteocalcin (OC) production (protein and RNA), and directly enhanced mineralization. 1Alpha,24R,25-(OH)3D3 stimulated ALP activity and OC production most potently, while for mineralization it was equipotent to 1alpha,25-(OH)2D3. The nuclear VDR antagonist ZK159222 almost completely blocked the effects of all metabolites. Interestingly, 1beta,25-(OH)2D3, an inhibitor of membrane effects of 1alpha,25-(OH)2D3 in the intestine, induced gene transcription and increased ALP activity, OC expression and mineralization. In conclusion, not only 1alpha,25-(OH)2D3, but also the presumed 24-hydroxylated "degradation" products stimulate differentiation of human osteoblasts. 1Alpha,25-(OH)2D3 as well as the 24-hydroxylated metabolites directly enhance mineralization, with the nuclear VDR playing a central role. The intestinal antagonist 1beta,25-(OH)2D3 acts in bone as an agonist and directly stimulates mineralization in a nuclear VDR-dependent way.
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