Estrogen modulates parathyroid hormone-induced fibronectin production in human and rat osteoblast-like cells.

Fibronectin (FN) is an important constituent of the extracellular matrix in bone. Its hormonal regulation in this tissue has not been extensively studied. We, therefore, examined the effects of PTH, estrogen, and transforming growth factor beta on the production of FN by human and rat osteoblast-like cells. Confluent cells were stabilized for 48 h under estrogen-replete (10(-9) M 17 beta-estradiol) conditions and then continued under these same conditions or withdrawn from estrogen for varying periods of time. PTH over the range 10(-11)-(10(-8) M caused a dose-dependent increase in FN production [P < 0.001 by analysis of variance (ANOVA)] such that at the highest dose, FN production was increased 11-fold. Estrogen withdrawal for 96 h caused a significant diminution in PTH-induced FN production (P < 0.005 by two-way ANOVA). Estrogen withdrawal over the of period 48-144 h caused a progressive diminution in PTH-induced FN production such that differences in mean values for estrogen-replete vs. deficient conditions were greater at 144 than 48 h (P < 0.05). The estrogen effect was titratable over the range 10(-11)-10(-9) M, and the inactive congener 17 alpha-estradiol failed to prevent the inhibitory effect of estrogen withdrawal on PTH-induced FN production. Interestingly, estrogen withdrawal had absolutely no effect on transforming growth factor-beta-induced FN production. Northern analysis demonstrated no effect of PTH on steady-state FN messenger RNA levels in Saos-2 cells under either estrogen-replete or estrogen-deficient conditions, suggesting that PTH effects an increase in FN production via a posttranscriptional mechanism in these cells. We conclude that PTH stimulates FN production in human and rat osteoblast-like cells, and under estrogen-deficient conditions this effect is significantly diminished. The modulatory effect of estrogen is not a universal phenomenon because transforming growth factor-beta-induced FN production is unaffected by estrogen withdrawal.

Parathyroid hormone increases circulating levels of fibronectin in vivo: modulating effect of ovariectomy.

To explore the effect of PTH on circulating levels of fibronectin (FN), adult female Sprague-Dawley rats were implanted with Alzet minipumps prepared to deliver 7 pmol/h x kg BW of either human PTH (1-34) or human PTH (1-84). Both forms of the hormone led to significant and progressive increases in circulating levels of FN over the 72-h study period (P < 0.001). However, at every time point, circulating levels of FN with human PTH (hPTH) (1-84) infusion were significantly higher than with hPTH (1-34), such that at the end of the infusion, mean levels in the hPTH (1-34) group were 32.2 +/- 1.4 ng/ml, in the hPTH (1-84) group 93.8 +/- 5.4 ng/ml, and in the vehicle infused group 14.6 +/- 0.7 ng/ml. The greater agonist efficacy of hPTH (1-84) was not explained by differences in circulating levels of the hormones, and both forms of the hormone were equipotent at stimulating cAMP production by ROS 17/2.8 cells. However, hPTH (1-84) remained a more effective agonist than hPTH (1-34) at stimulating FN production in these cells (P < 0.001). Nephrectomy did not blunt the ability of PTH to increase circulating FN in vivo, indicating that the kidney was not the source of the FN produced in response to PTH. Pretreament with the potent bisphosphonate APD to block bone resorption also did not blunt the in vivo response to PTH. Parathyroidectomy did not blunt the response. Cultured fetal rat bones showed a significant 2.4-fold increase in FN production when treated with PTH. Consistent with our earlier in vitro studies (Endocrinology, 135: 1639-1644, 1994), estrogen deficiency, induced by ovariectomy, significantly diminished the ability of PTH to increase circulating FN levels in vivo (P < 0.001). We conclude that PTH increases circulating levels of FN in vivo and may be a physiologic regulator for the plasma form of this glycoprotein. The effects of PTH on circulating FN may reflect the anabolic properties of the hormone in bone and the blunted response following estrogen withdrawal could be a manifestation of the diminished bone formation vis-à-vis resorption seen in the estrogen deficient state.

Tumor necrosis factor-alpha induces transcription of the colony-stimulating factor-1 gene in murine osteoblasts.

Tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption both in vitro and in vivo. The cellular mechanisms for this effect are not known but one pathway may be via release of osteoblast derived factors which stimulate osteoclast formation. Because colony-stimulating factor-1 (CSF-1) is essential for osteoclast progenitor proliferation, we examined the effect of TNF-alpha on osteoblast expression of CSF-1. TNF-alpha treatment of MC3T3-E1 or primary mouse osteoblasts stimulated the secretion of an activity that was mitogenic for a CSF-1 responsive cell line and was completely neutralized by antiserum to CSF-1. By Northern analysis, TNF-alpha caused a dose and time (3 to 24 h) dependent increase in CSF-1 transcript expression in MC3T3-E1 cells. mRNA stability studies using actinomycin D revealed that TNF-alpha does not affect CSF-1 mRNA half-life in MC3T3-E1 cells, while nuclear-run off analysis demonstrated that TNF-alpha increases CSF-1 gene transcription. Cycloheximide treatment of MC3T3-E1 cells up-regulated CSF-1 mRNA, and compared to either agent alone, cycloheximide and TNF-alpha in combination resulted in augmentation of CSF-1 expression. A series of studies using both agonists and inhibitors indicated that TNF-alpha-induced CSF-1 expression did not involve the arachidonic acid, PKC, or cAMP pathways. These results suggest that TNF-alpha induces CSF-1 expression in osteoblasts by a transcriptional mechanism which is largely independent of new protein synthesis and of the second messenger pathways examined.