Age-related changes in human oestrogen receptor alpha function and levels in osteoblasts.

Oestrogen receptors (ERs) are present in human osteoblasts and mediate anti-resorptive effects on bone. Human osteoblast-like cells derived from different aged healthy female donors not on hormone replacement therapy were utilized under well-defined conditions in vitro to investigate ER function and levels. Treatment with 0.1 nM oestradiol-17beta of cell strains derived from eight young women (less than 50 years of age) increased hydroxyproline levels significantly [an average (2.2+/-0.1 S.E.M.)-fold increase], whereas cells derived from nine older women (more than 50 years of age) were not significantly affected. Similarly, cell strains, derived from younger women, transfected with a consensus oestrogen-responsive element linked to chloramphenicol acetyltransferase exhibited a greater response to oestrogen than strains derived from older women. When basal ERalpha levels were measured by enzyme immunoassay and normalized on a per cell basis, osteoblast-like strains derived from younger women (n=24) had a mean value of 2.54+/-0.16 fmol of ERalpha per 10(6) cells. In contrast, strains derived from older women (n=20) had a mean value of 5.44+/-0.48 fmol of ERalpha per 10(6) cells. An age-related increase in ERalpha number was also observed in human skin-derived fibroblasts and directly in dermal biopsies from women not on hormone replacement therapy. The results demonstrate ligand concentration-dependent ERalpha induction and indicate a loss of receptor regulation and diminution of ligand-receptor signal transduction with increasing donor age.

Constitutive activation of fibroblast growth factor receptor 3 by mutations responsible for the lethal skeletal dysplasia thanatophoric dysplasia type I.

Thanatophoric dysplasia type I (TDI) is a neonatal lethal skeletal dysplasia caused by several mutations in the extracellular domain of fibroblast growth factor receptor 3. These mutations occur either in the Ig2-Ig3 linker domain or in the extracellular juxtamembrane domain, and all involve mutation of the wild-type residue to Cys. In all cases, the presence of the mutant Cys residue allows the receptor to dimerize abnormally, resulting in ligand-independent activation. This is also manifested by increased biological signaling, increased tyrosine phosphorylation, and in vitro kinase activity associated with dimeric receptors. These results suggest that TDI is caused by Cys-mediated intermolecular disulfide bonding, leading to constitutive receptor activation as a result of these mutations. Mutations causing TDI are discussed with respect to activating mutations in other receptors that are implicated in human disease.

Age-related changes in effects of insulin-like growth factor I on human osteoblast-like cells.

The role of insulin-like growth factor I (IGF-I) in extracellular matrix metabolism was studied in both proliferating and confluent human osteoblast-like cultures derived from donors of different ages. In proliferating cultures, recombinant human (rh)IGF-I was found to increase the incorporation of [3H]thymidine in a dose- and age-dependent manner. To study cell proliferation dynamically, continuous growth curves with and without rhIGF-I were modelled by a modified logistic function. Increasing doses of rhIGF-I decreased the lag time and maximal growth rates, whereas plateau values decreased only at the highest dose (100 ng/ml). In post-proliferative cell strains, rhIGF-I (0.1-100 ng/ml) increased levels of type I collagen, biglycan and decorin, and to a smaller extent fibronectin and thrombospondin, whereas it decreased the levels of hyaluronan and a versican-like proteoglycan when protein and proteoglycan metabolism were followed by steady-state radiolabelling with [3H]proline, [3H]glucosamine or [35S]sulphate. These responses to rhIGF-I were found to be age-dependent, with osteoblast-like cells derived from younger patients being more responsive to rhIGF-I. When extracellular matrix turnover was analysed by pulse-chase experiments, rhIGF-I had no effect. The steady-state levels of collagen, decorin, hyaluronan and a versican-like proteoglycan for bone cells treated with rhIGF-I on day 7 in culture were equivalent to levels of these matrix components in untreated osteoblasts grown for 14 days. These results are consistent with rhIGF-I's altering cellular proliferative capacity and matrix synthesis, causing a change in the osteoblast differentiated state.

Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II.

Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrent Lys-650-->Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the beta10-beta11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-650-->Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.

Cell proliferation of human fibroblasts and osteoblasts in osteogenesis imperfecta: influence of age.

Clinical studies indicate that as a group, osteogenesis imperfecta (OI) subjects are shorter than age- and sex-matched controls. Not only somatic growth, but also cellular growth appears to be impaired, and these may be related to defects in extracellular matrix common to this disorder. We have investigated the growth characteristics of dermal fibroblasts and trabecular osteoblasts isolated from patients with OI and control subjects of various ages. Cell growth curves and cell doubling times were determined by measuring cell number using crystal violet dye binding. Growth curves were modeled by a modified logistic function, the three parameters of which are markers for biologically relevant growth parameters: the plateau value or upper asymptote, which reflects the maximum cell density upon confluence; the maximal growth rate (microM); and the lag time. Both normal human fibroblasts and osteoblasts showed an age-dependent decrease in microM. Normal fibroblasts exhibited no age-dependence to their upper asymptote or lag time. Fibroblasts derived from patients with OI did not have significantly different upper asymptote values microM, or lag times when compared with normal fibroblasts. Normal osteoblasts had a decrease in upper asymptote, decrease in microM, but a relatively constant lag time with increasing age. In contrast, OI osteoblast microM was decreased relative to that of normal subjects. For osteoblasts from OI patients, decreased microM appeared unrelated to the age of the subject, whereas OI fibroblasts did exhibit an age-dependent decrease in microM. The percentage of collagenase-digestible protein (a measure of collagen synthesis) produced by normal human fibroblasts correlated well with microM. Treating normal human osteoblasts with the proline analogue 3,4-dehydroproline, which destabilizes collagen triple helix formation and alters collagen synthesis, secretion, and turnover, also decreased microM. A dose response to varying concentrations of 3,4-dehydroproline was observed for normal human bone cell microM. These data suggest a link between type I collagen synthesis and cellular proliferation.