Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor-beta1 (TGF-beta1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF-beta1-induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF-beta1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF-beta1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF-specific siRNA was used to prevent TGF-beta1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the requirement of CTGF for their up-regulation. To examine the effects of TGF-beta1 on osteoblast cell growth, cultures were treated with TGF-beta1 during the proliferative stage. Cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression, consistent with TGF-beta1-induced cell-cycle arrest. Cultures transfected with CTGF siRNA prior to TGF-beta1 treatment showed an even greater reduction in cell number, suggesting that TGF-beta1-induced growth arrest is independent of CTGF in osteoblasts. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF-beta1-induced ECM production in osteoblasts, but these two growth factors function independently regarding their opposing effects on osteoblast proliferation.

Cig30 and Pitx3 genes are arranged in a partially overlapping tail-to-tail array resulting in complementary transcripts.

The mouse Cig30 gene codes for a 30-kDa membrane glycoprotein, which appears to have a role in the recruitment of brown adipose tissue. To elucidate the structure of the Cig30 gene, we have isolated a lambda phage genomic DNA clone containing the entire mouse gene and found that Cig30 consists of four exons that are spread over 4 kilobase pairs of genomic sequence. Using a fluorescence in situ hybridization assay and interspecific backcross panel mapping, we have localized the Cig30 locus to the distal region of mouse chromosome 19, between the Tlx1 and Ins1 loci. Sequencing of the corresponding lambda clone to completion revealed that the insert contained yet another gene in the opposite orientation. It turned out to be the newly identified homeobox gene Pitx3. Interestingly, the genes are very tightly linked, so that the 3' ends of their transcripts are complementary. Thus, our results provide evidence for bidirectional transcription of a several hundred base pair-long DNA region as a result of the extremely tight linkage between Cig30 and Pitx3.

Gene expression of natriuretic peptide receptors in rats with DOCA-salt hypertension.

In our previous studies, we found that the atrial natriuretic peptide (ANP) binding and guanylyl cyclase activity of A-type natriuretic peptide receptors (NPR-A) were upregulated in renal papillae but downregulated in vascular tissues and glomeruli of rats with deoxycorticosterone acetate (DOCA)-salt hypertension [E. Nuglozeh, G. Gauquelin, R. Garcia, J. Tremblay, and E. L. Schiffrin. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F130-F137, 1990]. To further understand the molecular significance of these regulations, we measured the relative abundance of the transcripts of NPR-A and NPR-B by Northern blot in the aorta, mesenteric arteries, adrenal cortex, renal papillae, and lungs in DOCA-salt hypertensive and control rats. In renal papillae we also examined the translation and transcription of NPR-A by ribosome loading and run-on assay. Compared with controls, the steady-state levels of mRNA for NPR-A were increased in the aorta and mesenteric arteries but were decreased in the adrenal cortex and renal papillae in DOCA-salt-treated rats. NPR-B mRNA was decreased in the aorta, mesenteric arteries, and adrenal cortex in hypertensive rats. In lungs the mRNA for both receptors was unchanged. Translation of NPR-A mRNA, as assessed by ribosome loading, was reduced in renal papillae. Transcriptional activity of its gene was not detectable in these tissues. Guanosine 3',5'-cyclic monophosphate levels generated by NPR-A in renal papillae and by NPR-A and NPR-B in the adrenal cortex, aorta, and mesenteric arteries of DOCA-salt-treated rats remained increased in hypertension. The higher NPR-A activity in the presence of a lower level of its mRNA in renal papillae and the higher NPR-B activity in the presence of a lower level of its mRNA in the vasculature, adrenal cortex, and lungs can alternatively be explained by receptor stabilization or increased receptor recycling.

Dietary calcium supplementation and dopamine-beta-hydroxylase in spontaneously hypertensive rats.

Spontaneously hypertensive 4-week-old male rats were fed, before and after the onset of hypertension, with either commercial chow (control) or commercial chow combined with different forms of milk proteins with or without calcium supplementation. After 40 weeks, rats were still hypertensive, and dopamine-beta-hydroxylase enzyme activity measured simultaneously in serum and adrenal was found to be higher than in the controls. The enzyme activity in rats fed diets with milk proteins was increased significantly in both serum and adrenal compared with the control, and such enhancement was significantly higher than that observed in animals fed the commercial diet supplemented with calcium (1.2%), suggesting that dietary calcium intake associated with dietary protein of high digestibility, such as casein, potentiates the endogenous mechanisms regulating the homeostasis of calcium more than calcium supplementation itself. Moreover, the selective and additive effect of diets supplemented with milk proteins and calcium on adrenal enzyme activity clearly suggests a relationship between cardiovascular diseases involving the genesis of hypertension and stress mechanisms through the hypothalamo-pituitary adreno-sympathetic axis.

Rat natriuretic peptide receptor genes are regulated by glucocorticoids in vitro.

The effects of glucocorticoids on NPR-A and NPR-B mRNA transcription and natriuretic peptides ANP and CNP mediated cGMP production by intact vascular smooth muscle cells (VSMC) were studied in rat. Cultured VSMC were prepared from rat mesenteric arteries of 12-week-old Sprague-Dawley rats by enzymatic digestion. Dexamethasone-induced NPR-A mRNA increase was detectable early in the incubation periods and reached a plateau after 48 hours of glucocorticoid administration. This mRNA increase was mimicked by cortisol and inhibited by the glucocorticoid receptors antagonist RU 38,486. The levels of NPR-B mRNA remained unchanged during all the periods of stimulation. However, cGMP generated by both receptors in dexamethasone treated cells was higher than in control cells and this production was mimicked by cortisol and also blocked by RU 38,486. Desoxycorticosterone acetate (DOCA) had no effect on the levels of cGMP production. The results suggest that glucocorticoids have transcriptional and posttranscriptional effects on rat mesenteric arteries cells through glucocorticoid receptors.

Atrial natriuretic peptide receptors in renal papilla of DOCA-salt hypertensive rats.

The receptor for atrial natriuretic peptide (ANP) in the rat renal papilla was characterized pharmacologically. After solubilization and irreversible binding with disuccinimidylsuberate, it was shown on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to be made of a single peptide of 125 kDa. The regulation of the renal papillary ANP receptor was studied in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. DOCA-salt rats had suppressed plasma renin activity and increased plasma ANP concentrations (408 +/- 35 vs. 133 +/- 12 pg/ml in uninephrectomized controls, P less than 0.01). The renal papilla was hypertrophied in DOCA-salt hypertensive rats (93 +/- 1 vs. 52 +/- 1 mg, P less than 0.01). The density of ANP sites in the papilla was significantly higher in DOCA-salt rats (141 +/- 31 fmol/papilla) than in controls (34 +/- 8 fmol/papilla, P less than 0.01). Affinity of sites in DOCA-salt rats and controls was similar. The production of guanosine 3',5'-cyclic monophosphate (cGMP) in renal papilla in response to ANP was significantly higher in DOCA-salt rats. In contrast to the renal papillary ANP receptor, acid-washed vascular and glomerular ANP sites were significantly decreased in density in DOCA-salt hypertensive rats. In blood vessels and glomeruli, both the high- and low-molecular mass receptor (as detected on SDS-PAGE under reducing conditions) was proportionately decreased in density in DOCA-salt hypertensive rats. The present results suggest that an increased number of ANP receptors and exaggerated cGMP response to ANP in the renal papilla may underlie the increased natriuretic responsiveness of the kidney to ANP in DOCA-salt hypertensive rats.