PTHrP expression in chick sternal chondrocytes is regulated by TGF-beta through Smad-mediated signaling.

PTHrP regulates the rate of chondrocyte differentiation during endochondral bone formation. The expression of PTHrP and its regulation by TGF-beta, BMP-2, and PTHrP was examined in upper sternal chondrocytes following 1, 3, and 5 days of continuous treatment. While TGF-beta stimulated the expression of PTHrP (5-fold), PTHrP caused a slight inhibition, and BMP-2 markedly inhibited PTHrP mRNA expression. The effect of these factors on PTHrP expression was not simply related to the maturational state of the cells, since BMP-2 increased, while both PTHrP and TGF-beta decreased the expression of type X collagen. TGF-beta isoforms 1, 2, and 3 all stimulated PTHrP expression. Signaling events involved in the induction of PTHrP by TGF-beta were further evaluated in a PTHrP-promoter CAT construct. The effect of TGF-beta, BMP-2, and PTHrP on the PTHrP-promoter paralleled their effects on mRNA expression, with TGF-beta significantly increasing CAT activity, BMP-2 decreasing CAT activity, and PTHrP having a minimal effect. Co-transfection of the TGF-beta signaling molecule, Smad 3, mimicked the effect of TGF-beta (induction of PTHrP promoter), while dominant negative Smad 3 inhibited the induction of the PTHrP promoter by TGF-beta. Furthermore, infection with a Smad 3-expressing retrovirus mimicked the effects of exogenously added TGF-beta, and induced PTHrP mRNA expression in the infected chondrocyte culture. In contrast, a dominant negative Smad 3 completely inhibited PTHrP promoter stimulation by TGF-beta, but only partially blocked the effect of TGF-beta on PTHrP mRNA synthesis. These findings demonstrate that PTHrP is expressed in chondrocytes undergoing endochondral ossification, and show regulation, at least in part, by TGF-beta through Smad mediated signaling events.

The role of autocrine growth factors in radiation damage to the epiphyseal growth plate.

Radiation therapy plays an important role as part of the multimodality treatment for a number of childhood malignancies. Dose-limiting complications of radiotherapy include skeletal abnormalities and disturbances in skeletal development within the irradiated field. The current study was undertaken to investigate the molecular mechanisms involved in radiation-induced arrest of bone growth. Our hypotheses were: (1) Expression of autocrine growth factors that regulate chondrocyte proliferation is inhibited by radiation in a specific pattern; (2) the disparity in radiosensitivity of growth plate chondrocytes and epiphyseal chondrocytes is due to differential modulation of autocrine growth factor expression by radiation. Given the important role these cells play in skeletal growth and development, we examined the comparative effects of radiation on expression of specific mitogenic growth factors in growth plate chondrocytes. The effect of radiation on the expression of autocrine/paracrine growth factors was examined in an established avian model of epiphyseal growth plate maturation. Exposure of growth plate chondrocytes to radiation resulted in a specific pattern of biochemical and morphological alterations that were dependent on dose and were progressive over time. While radiation did not affect the mRNA expression of some of the autocrine and paracrine factors important in endochondral ossification (such as FGF2 and TGFB isoforms), it did lead to a decrease in the mRNA expression of PTHrP, a critically important mitogen in growth plate chondrocytes, and a dose-dependent decrease in the PTH/PTHrP receptor mRNA. Interestingly, PTHrP mRNA levels were not affected in irradiated epiphyseal chondrocytes, the main source of PTHrP. Given evidence indicating a role for intracellular calcium levels in regulating PTHrP expression, basal calcium levels in irradiated growth plate chondrocytes and epiphyseal chondrocytes were examined 24 h after treatment. While cytosolic calcium levels were significantly higher in irradiated growth plate chondrocytes, they were not significantly affected in irradiated epiphyseal chondrocytes. The importance of calcium in mediating radiation damage to growth plate chondrocytes was further demonstrated by the finding that the addition of 4.0 mM EGTA (a calcium chelator) to the cell cultures before irradiation prevented the decrease in PTHrP mRNA levels. Since PTHrP up-regulates BCL2 levels and prevents growth plate chondrocyte maturation and apoptosis, BCL2 mRNA levels were examined in irradiated growth plate chondrocytes, and a dose-dependent decrease was found. An increase in apoptosis was further confirmed by a fivefold increase in caspase 3 levels in irradiated growth plate chondrocytes. The results of the current study suggest that radiation may interfere with proliferation of growth plate chondrocytes in part by causing an increase in cytosolic calcium levels which in turn leads to a decrease in PTHrP mRNA. Growth plate chondrocyte PTHrP receptor mRNA expression is also inhibited by radiation, further decreasing PTHrP signaling. Despite subtle differences between the chick and mammalian growth plates, further studies should provide an enhanced understanding of the mechanism(s) of radiation injury to the growth plate, as well as possibilities for new therapeutic strategies to protect the growing skeleton from the detrimental effects of radiotherapy.

PTHrP expression in chondrocytes, regulation by TGF-beta, and interactions between epiphyseal and growth plate chondrocytes.

Although PTHrP has been identified as a key regulator of chondrocyte differentiation in the growth plate, the factors directly regulating PTHrP expression have not been identified. Furthermore, while cells from the epiphysis are considered the physiologic source of PTHrP, the relative expression of PTHrP in epiphyseal and growth plate chondrocytes has not been defined. PTHrP expression was examined in chondrocytes isolated from 3- to 5-week-old chick long bones. The expression of PTHrP mRNA was 10-fold higher in epiphyseal chondrocytes compared to cells from the growth plate. Growth plate chondrocytes were isolated into populations with distinct maturational characteristics by countercurrent centrifugal elutriation and analyzed for PTHrP expression. The expression was highest in the least mature cells and progressively declined with the onset of maturation. The regulation of PTHrP expression was further examined in epiphyseal chondrocytes. Both TGF-beta1 and cis-retinoic acid stimulation markedly increased PTHrP mRNA levels, while BMP-2 and PTHrP stimulation decreased the expression of this transcript. The effects of TGF-beta1 (8.9-fold stimulation) and TGF-beta3 (9.2-fold) were slightly greater than the effects of TGF-beta2 (4.9-fold). The effect of TGF-beta was dose-dependent and increases could be detected after 68 h of treatment. To analyze the paracrine effect of epiphyseal and growth plate chondrocytes on each other, these cells were placed in coculture and the mRNA from each of the populations was harvested separately after 24 h. Following coculture the PTHrP mRNA levels increased in the epiphyseal cells while the expression of type X collagen and Indian hedgehog transcripts decreased in growth plate chondrocytes. The results demonstrate potentially important paracrine interactions between these cell populations, possibly mediated by TGF-beta and PTHrP.