Isolation, differential splicing and protein expression of a DNase on the human X chromosome.

A systematic search for genes differentially expressed in human tissues resulted in the isolation of a gene encoding a protein with high homology to DNase I. In addition to the recently described cDNA sequence (Parrish et al., 1995) we have isolated a transcript, alternatively spliced in the 5' noncoding region. The gene is located between the QM and the XAP-2 gene in Xq28 and encodes a 302 amino acid protein with 39% identity to human DNase I. Besides a high homology at the nucleotide and amino acid level, most exon-intron boundaries of DNase I and DNase X are identical, indicating that both genes may have evolved from a common ancestor. The predicted function was verified by expression of a recombinant protein in an inducible bacterial system and detection of DNase activity. In contrast to DNase I a 18 kdal amino terminal fragment of the full length 35 kdal protein exhibited DNase activity.

Vitamin D and dexamethasone inversely regulate parathyroid hormone-induced regulator of G protein signaling-2 expression in osteoblast-like cells.

The PTH/PTHrP receptor stimulates both adenylate cyclase- and phospholipase C-dependent signaling pathways via different G proteins. The biological actions of PTH on bone are modified by steroid hormones. PTH induces expression of regulator of G protein signaling (RGS)-2, a putative preferential inhibitor of G(q)-mediated phospholipase C activation. We investigated whether steroid hormones interfere with PTH signaling by modulating PTH-induced RGS-2 expression in osteoblast-like UMR 106-01 cells. PTH (1-34) rapidly and transiently induced expression of RGS-2 mRNA and protein via the cAMP/protein kinase A pathway within 30 min, with maximal protein abundance after 2 h. PTH-induced RGS-2 preferentially bound to Galpha(q), compared with Galpha(s) protein. 1,25-(OH)(2)D(3) pretreatment enhanced PTH-induced RGS-2 mRNA and protein accumulation, whereas dexamethasone preincubation had an attenuating effect. These effects were due to modulation of the RGS-2 gene transcription rate, which increased by 35% with 1,25-(OH)(2)D(3) and decreased by 63% with dexamethasone pretreatment. RGS-2 mRNA half-life was not affected by either steroid. The transcriptional effects of dexamethasone and 1,25-(OH)(2)D(3) were independent of PTH/PTHrP receptor activation and were not explained by effects on cAMP accumulation, cAMP response element-binding protein expression or phosphorylation, or the abundance of the osteoblast-specific transcription factor core-binding factor alpha (CBFa1/Runx2), a known activator of RGS-2 expression. In conclusion, glucocorticoids and 1,25-(OH)(2)D(3) inversely modulate PTH-induced RGS-2 gene transcription. Regulation of RGS-2 may constitute a novel mechanism by which steroids modulate signaling via the PTH/PTHrP receptor and other G protein-coupled receptors in bone.

The interaction of glucocorticoids with the growth hormone-insulin-like growth factor axis and its effects on growth plate chondrocytes and bone cells.

Glucocorticosteroids interfere with the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis at different levels, and while low-dose corticosteroids may have permissive effects, high-dose, long-term treatment with corticosteroids may lead to growth disturbance. The mechanism involved is not clearly understood. The Janus kinase (JAK)-2/signal transducers and activators of transcription (STAT)-5 pathway is the means by which the corticosteroid interacts with the target-cell GH receptors. The production of local IGF-I is lowered by the corticosteroid via IGF-I transcription inhibition, and the rate of apoptosis is also increased, both in growth plate chondrocytes and osteoblast cell lines. GH in vitro and in vivo can partly counterbalance the negative effects of glucocorticoids on growth. GH has been seen to normalize growth rates in corticosteroid-treated rats as well as in children receiving glucocorticoids for immunosuppression following kidney transplantation.

Suppression of growth plate chondrocyte proliferation by corticosteroids.

Growth depression is a side effect of high-dose glucocorticoid therapy in childhood. It is partially mediated by alterations of the somatotropic hormone axis and partially by direct local effects on growth plate chondrocytes. The mechanisms of interaction of corticosteroids and somatotropic and calciotropic hormones at the cellular level were recently investigated in more detail, using experimental models of primary cultures of growth plate chondrocytes. In proliferative chondrocytes, growth hormone (GH) and the calciotropic hormones parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1alpha,25(OH)2D3] increase cell proliferation via stimulation of paracrine insulin-like growth factor-I (IGF-I) secretion. Corticosteroids decreased GH, and PTH or 1alpha,25(OH)2D3 stimulated cell growth in a dose-dependent manner. Corticosteroids in high doses reduced the expression of the GH receptor and type 1 IGF receptor. But the main antiproliferative molecular effect of corticosteroid was the reduction in basal and hormone-stimulated IGF-I secretion. The in vitro results are in accordance with the observation in animal experiments and in children treated with corticosteroids, demonstrating that the growth-depressing effect of corticosteroids can be compensated for by supraphysiological doses of GH or IGF-I.

1,25(OH)2D3 and dihydrotestosterone interact to regulate proliferation and differentiation of epiphyseal chondrocytes.

Growth plate chondrocytes are affected by 1,25(OH)2D3 and androgens, which may critically interact to regulate proliferation and differentiation during the male pubertal growth spurt. We investigated possible interactions of 1,25(OH)2D3 and the non-aromatizable androgen dihydrotestosterone (DHT) in primary chondrocyte cultures from young male rats. DHT and 1,25(OH)2D3 independently stimulated DNA synthesis and cell proliferation in a dose-dependent manner with maximally effective doses of [10(-8) M] and [10(-12) M], respectively. Both DHT and 1,25(OH)2D3 stimulated the expression and release of IGF-I, and the proliferative effects of each hormone were prevented by an IGF-I antibody. DHT and 1,25(OH)2D3 increased messenger RNAs (mRNAs) of their cognate receptors and of IGF-I receptor mRNA (IGF-I-R). 1,25(OH)2D3 also stimulated mRNA of the androgen receptor (AR), whereas DHT did not affect mRNA of the vitamin-D receptor (VDR). Coincubation with both steroid hormones did not stimulate receptor mRNAs more than either hormone alone. The proliferative effects of DHT and 1,25(OH)2D3 were completely inhibited by simultaneous incubation with both hormones, despite potentiation of IGF-I synthesis. In contrast, both hormones synergistically stimulated cell differentiation as judged by alkaline phosphatase activity, collagen X mRNA, and matrix calcification in long-term experiments. We conclude that DHT and 1,25(OH)2D3 interact with respect to chondrocyte proliferation and cell differentiation. The proliferative effects of both hormones are mediated by local IGF-I synthesis. Simultaneous coincubation with both hormones blunts the proliferative effect exerted by either hormone alone, in favor of a more marked stimulation of cell differentiation.