Synergistic effect of transforming growth factor beta and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes.

Transforming growth factor beta and fibroblast growth factor are mitogens for chick growth plate chondrocytes. TGF-beta stimulated a 3.5-fold increase, and FGF a 13.5-fold increase in the rate of thymidine incorporation after a 24 h exposure. TGF-beta and FGF were synergistic in chondrocytes, causing a 73-fold stimulation in thymidine incorporation compared with control. This synergistic response was not dependent upon the simultaneous presence of both mitogens. Sequential exposure of chondrocytes to TGF-beta and FGF in either order reproduced in large part the synergistic interaction observed when both growth factors were present simultaneously. The time required for induction of the subsequent synergistic response was brief and, in the case of TGF-beta, corresponded to the time required for [125I]TGF-beta receptor binding. EGF and PDGF were not mitogenic for chondrocytes, and neither of these factors enhanced the response of the cells to either TGF-beta or FGF. Finally, TGF-beta and FGF did not, either alone or in combination, elevate intracellular cAMP levels. These results emphasize the importance of examining growth factor effects in the context of other growth regulators. Furthermore, this specific and dramatic synergistic stimulation of thymidine incorporation may provide a useful tool in elucidating the mitogenic mechanism of the individual growth factors.

Influence of prostaglandins on DNA and matrix synthesis in growth plate chondrocytes.

Prostaglandins are locally produced in a number of tissues in response to a variety of stimuli, including local growth factors and systemic hormones. The present investigation characterizes prostaglandin effects on growth plate chondrocytes. Since cyclic adenosine monophosphate (cAMP) may act as a prostaglandin-stimulated second messenger, the effects of prostaglandins A1, D2, E1, E2, F2 alpha, and I2 (10(-10)-10(-6) M) on cAMP levels and thymidine incorporation were evaluated. The stimulation of cAMP and thymidine incorporation by the various prostaglandin metabolites were dose dependent and highly correlated (r = 0.99, p less than 0.001). The magnitude of the effect varied but was maximal at 10(-6) M for each of the prostaglandins. Prostaglandins of the E series (E1 and E2) were the most potent, causing significant effects at 10(-10) M and with maximal 12- and 13-fold increases in DNA synthesis after a 24 h exposure. Prostaglandins D2 and A1 maximally stimulated thymidine incorporation by 4.7- and 3.1-fold but caused significant increases only at 10(-8) M. Prostaglandins F2 alpha and I2 were the least stimulatory, producing small but significant increases in thymidine incorporation at 10(-6) M (30 and 100% stimulations). A causal relationship between cAMP and thymidine incorporation was further verified by the ability of dibutyryl-cAMP to increase DNA synthesis. Long-term chondrocyte cultures treated continuously with PGE2 demonstrated an increase in cell number, confirming the proliferative effect. Indomethacin did not alter the potent dose-dependent stimulations of chondrocyte DNA synthesis by TGF-beta 1, basic FGF, or PTH, indicating that these known mitogens act independently of prostaglandin metabolism. PGE2 was further examined for its effects of matrix synthesis. PGE2 inhibited collagen synthesis with a maximal 42% decrease but did not alter noncollagen protein synthesis. In contrast, PGE2 maximally increased sulfate incorporation by 35% and caused a small dose-dependent inhibition in alkaline phosphatase activity. Thus, prostaglandins alter DNA and matrix synthesis in growth plate chondrocytes and may have an important role in chondrocyte metabolism in the growth plate, fracture callus, and other areas of endochondral ossification.

Shared phenotypic expression of osteoblasts and chondrocytes in fracture callus.

Endochondral ossification in fracture healing of rats at 4, 8, 11, 14, and 21 days was analyzed using immunological and molecular probes for markers of the chondrocyte and osteoblast phenotype. These markers were osteocalcin, type I and type II collagen, including the probes homologous to the alternatively spliced forms of alpha 1 type II collagen, type IIA and type IIB. Histologic examination was performed on serial sections of the same tissue blocks to correlate cellular morphology with the immunohistochemical and in situ hybridization findings. At the junction of the cartilaginous and osseous tissue, an overlap of phenotype and morphology was noted. At the 8-day time point, the cells with chondrocyte morphology expressed intracellular message for osteocalcin and type I collagen. Immunohistochemical analysis of these cells also demonstrated intracellular osteocalcin. However, high levels of the type IIA collagen mRNA, which has previously been associated with less differentiated mesenchymal precursor cells, were expressed in both chondrocytes and osteoblasts. At the later time point (21 days) there was a substantial decrease in the number of cells displaying shared phenotypic characteristics. In situ hybridization and immunohistochemistry have permitted identification of an overlapping or shared phenotype in osteoblasts and chondroblasts in fracture callus. The findings raise important questions regarding the possible plasticity of mesenchymal cell phenotypes within the dynamic environment of fracture healing. Additional examination of these issues will further define factors involved in origin, differentiation, and maturation of bone and cartilage cells.

Effects of transforming growth factor-beta 1 and fibroblast growth factor on DNA synthesis in growth plate chondrocytes are enhanced by insulin-like growth factor-I.

The local tissue metabolism is controlled through the complex interaction between systemic and local growth factors. In recent years, an increasing number of autocrine or paracrine growth regulators have been identified in physeal cartilage. While these factors act to alter chondrocytes phenotypically and presumably are important mediators in the process of endochondral ossification, the manner in which they interact with the systemically regulated growth factor insulin-like growth factor-I is unknown. In the present study, the interactive effects of insulin-like growth factor-I with transforming growth factor-beta 1 or basic fibroblast growth factor were examined in short-term monolayer cultures of chick growth plate chondrocytes. [3H]thymidine incorporation was maximally stimulated 11-fold by fibroblast growth factor (10 ng/ml) and 3.5-fold by transforming growth factor-beta 1 following a 24-hour exposure in serum-containing cultures. The effects of transforming growth factor-beta 1 and fibroblast growth factor at both high and low concentrations were enhanced in a dose-dependent manner by insulin-like growth factor-I, with a 40-50% increase in DNA synthesis in the presence of 100 ng/ml of insulin-like growth factor-I. Since insulin-like growth factor-I increased [3H]thymidine incorporation after 48 hours (50% increase) but not after 24 hours of exposure, these observations represent a synergistic interaction. Total DNA in cultures treated for 5 days confirmed the modulating effect of insulin-like growth factor-I with transforming growth factor-beta 1 and fibroblast growth factor. The growth factors were further examined for their effects on markers of chondrocyte differentiation. While all three caused a dose-dependent inhibition of alkaline phosphatase activity, the effects of insulin-like growth factor-I were additive only to those of transforming growth factor-beta 1 and fibroblast growth factor. Similarly, insulin-like growth factor-I did not affect the sulfate incorporation stimulated by fibroblast growth factor or transforming growth factor-beta 1. Insulin-like growth factor-I had no effect on total protein synthesis after 24 hours and, although type-II collagen mRNA levels were stimulated, it had no effect on type-X collagen mRNA, as determined by quantitative in situ hybridization. Finally, insulin-like growth factor-I did not alter the dose-dependent stimulation of noncollagen protein synthesis and the inhibition of collagen synthesis caused by fibroblast growth factor and transforming growth factor-beta 1 in 24-hour cultures. Thus, the data suggest that insulin-like growth factor-I may have a role in augmenting the effects of other growth factors found in cartilage.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential regulation of type-II and type-X collagen synthesis by parathyroid hormone-related protein in chick growth-plate chondrocytes.

Parathyroid hormone-related protein is a critical autocrine regulator of endochondral ossification in the growth plate, as demonstrated by the severe disruption of growth-plate structure and function in parathyroid hormone-related protein-deficient transgenic mice. In the present study, the effects of parathyroid hormone-related protein on the synthesis of collagen mRNA and protein were studied in short-term cultures of isolated chick growth-plate chondrocytes. Parathyroid hormone-related protein selectively inhibits type-X collagen protein synthesis with no significant effect on type-II collagen protein synthesis. These effects were present in all maturationally distinct populations of chondrocytes separated by countercurrent centrifugal elutriation. In cultures of resting chondrocytes, the onset of type-X collagen expression was inhibited, while the synthesis of type-X collagen was decreased in cultures of hypertrophic chondrocytes. Synthesis of type-II and type-X collagen mRNA was examined by nonradioactive in situ hybridization with synthetic oligonucleotide cDNA probes, and the level of expression was quantified using digital image analysis. Dose-dependent suppression of type-X collagen gene expression by parathyroid hormone-related protein was observed, with no significant effect on type-II collagen mRNA detected. The results were confirmed by analysis of Northern blots of total chondrocyte mRNA. These experiments demonstrated differential transcriptional regulation of type-II and type-X collagen, with selective suppression of type-X collagen expression, by parathyroid hormone-related protein in growth-plate chondrocytes. In addition, excellent agreement was found between traditional protein and mRNA analyses and microscopic digital image analysis techniques, supporting the use of this convenient and sensitive assay method. Parathyroid hormone-related protein inhibits chondrocyte maturation and is known to stimulate proliferation, suggesting that this autocrine factor may function to regulate premature hypertrophy in the growth plate.

Countercurrent centrifugal elutriation. High-resolution method for the separation of growth-plate chondrocytes.

Countercurrent centrifugal elutriation was used to separate, on the basis of size, isolated growth-plate chondrocytes in chicks. The mean cellular volume, activity of alkaline phosphatase, and synthesis of type-X collagen increased progressively in each of seven successive fractions. Slices of tissues that contained either proliferating or hypertrophic chondrocytes were also removed by manual dissection from the superficial and deep regions of the growth plate. These cells demonstrated differences in size and biochemistry that were similar to those observed in chondrocytes that were separated by elutriation. These differences included increased synthesis of proteoglycan and collagen in the larger chondrocytes. Radiolabeled hypertrophic chondrocytes were mixed with unlabeled resting and proliferating chondrocytes, and then were separated by elutriation. The radioactivity was recovered in fractions that contained the largest cells, confirming that differences in the sizes of the cells can be used to effect a zonal separation by centrifugal elutriation.

Transforming growth factor beta: an autocrine regulator of chondrocytes.

Transforming growth factor beta (TGF-beta) is a ubiquitous regulator of cellular growth and differentiation. The present study investigated the effects of TGF-beta on chick growth plate chondrocyte proliferation, matrix synthesis, and alkaline phosphatase activity in short term cultures. TGF-beta markedly stimulated DNA synthesis in a dose-dependent manner, while collagen synthesis and cellular and matrix vesicle alkaline phosphatase activity were inhibited. Biologic effects of TGF-beta were correlated with binding to specific receptors, and both high and low affinity receptors were identified. Countercurrent centrifugal elutriation was used to fractionate growth plate chondrocytes to obtain populations of cells in different stages of maturation (effectively from different zones of the growth plate). TGF-beta showed increasing mitogenicity with increasing cellular maturation in the growth plate, with maximal stimulation in the proliferating and early hypertrophic cells. The smallest cells expressed only the high affinity receptor, while with hypertrophy there was increasing expression of the low affinity receptor and a progressive increase in the number of both receptors per cell. Furthermore, the dose-response curves for TGF-beta-stimulated DNA synthesis were not biphasic in the smaller cells, but became progressively more biphasic with cellular hypertrophy and expression of the low affinity receptor. Finally, TGF-beta activity was identified in partially purified chondrocyte conditioned medium by specific bioassay, indicating TGF-beta production by growth plate chondrocytes. The data suggests a potentially important autocrine function for TGF-beta in modulating chondrocyte proliferation and matrix synthesis in endochondral calcification.

Differential effects of parathyroid hormone on chick growth plate and articular chondrocytes.

Parathyroid hormone (PTH) binds specifically to the hypertrophic region of growth plate cartilage [16]. This specific binding suggests a role for this hormone in chondrocyte maturation. Enzymatically isolated chick articular and growth plate chondrocytes grown in monolayer culture were used to assay the direct effects of PTH on chondrocytes. The articular chondrocytes were unresponsive to PTH. The growth plate chondrocytes, however, demonstrated a marked mitogenic response to PTH, with a 39-fold increase of [3H]-thymidine incorporation into DNA. PTH also affected matrix production by the growth plate chondrocytes causing a twofold stimulation of proteoglycan synthesis as determined by the rate of 35SO4 incorporated into matrix macromolecules. Furthermore, PTH depressed collagen synthesis as measured by [3H]-proline incorporation. PTH caused a 12-fold increase in intracellular cAMP in growth plate chondrocytes but no increase in the articular cells. This specificity of PTH for growth plate chondrocytes suggests a possible regulatory role in enchondral ossification.

Nonradioactive in situ hybridization using digoxigenin-labeled oligonucleotides. Applications to musculoskeletal tissues.

We have optimized a technique for in situ localization of specific mRNAs using digoxigenin-11-dUTP-labeled oligonucleotide probes. DNA probes were synthesized for type I and type II collagen as well as transforming growth factor-beta 1 and 2 (TGF beta 1 and TGF beta 2). Control experiments, such as competitive inhibition, nonsense sequence hybridization, and RNAse digestion all indicated that the technique was highly sensitive and specific. In sections of growth plate, type II collagen mRNA was predominantly expressed in the lower proliferative and upper hypertrophic zone, whereas chondrocytes in articular cartilage stained equally. These techniques then were applied to sections cut from archival pathology specimens of musculoskeletal tissues. Primitive chondrocytes in a chondrosarcoma expressed type I and type II collagen mRNA, but did not stain with the nonsense probe. Sections from an osteosarcoma, an aneurysmal bone cyst, and a neurofibroma also were investigated. The ability to use chemically synthesized oligonucleotide probes, the high resolution, and the short development times possible with this in situ procedure makes this technique appealing for applied research into the gene expression of normal and pathologic cellular events.