RESUMEN
We have used in situ hybridization to evaluate the effects of 1,25 dihydroxyvitamin D3 (1,25 (OH)2 D3) on the expression of mRNA for bone-matrix proteins and to determine whether mature osteoblasts respond differently to 1,25 (OH)2 D3 than younger, newly differentiated osteoblasts. Rat calvaria cells were cultured for 7, 12, 15, and 19 days to obtain a range of nodules from very young to very mature. At each time point, some cultures were treated with 10 nM 1,25 (OH)2 D3 for 24 h prior to fixation. In control cultures, type-I collagen mRNA was detectable in osteoblastic cells in very young nodules and increased with increasing maturity of the nodules and the osteoblasts lining them. The bone sialoprotein mRNA signal was weak in young osteoblasts, increased in older osteoblasts, and decreased in mature osteoblasts. Weak osteocalcin and osteopontin signals were seen only in osteoblasts of intermediate and mature nodules. 1,25 (OH)2 D3 treatment markedly upregulated osteocalcin and osteopontin mRNAs and downregulated mRNA levels of bone sialoprotein and, to a lesser extent, type-I collagen in both young and mature osteoblasts. However, a marked diversity of signal levels for bone sialoprotein, osteocalcin, and osteopontin existed between neighboring mature osteoblasts, particularly after 1,25 (OH)2 D3 treatment, which may therefore selectively affect mature osteoblasts, depending on their differentiation status or functional stage of activity.
Asunto(s)
Calcitriol/fisiología , Colágeno/genética , Osteoblastos/metabolismo , Osteocalcina/genética , ARN Mensajero/biosíntesis , Sialoglicoproteínas/genética , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Sialoproteína de Unión a Integrina , Osteoblastos/citología , Osteopontina , Ratas , Ratas WistarRESUMEN
We investigated whether the expressed phenotype of osteoblasts and odontoblasts is changing with increasing maturity of the cells. Thus we determined, using in situ hybridization techniques, whether the expression of mRNA's for osteocalcin and the alpha 1 type 1 collagen chain was different in newly developed and more mature cuboidal osteoblasts of the primary and secondary spongiosa of radii of 8 day old rats, in mature cuboidal and older flat osteoblasts in the metatarsals of pig embryos, and in apical and coronal odontoblasts of the developing unerupted molars of pig embryos. The results indicate that newly differentiated osteoblasts in the primary spongiosa of the 8 day rat radius contained approximately the same amount of type 1 collagen message as more mature osteoblasts in the secondary spongiosa. Osteocalcin mRNA, on the other hand, was undetectable in the newly differentiated osteoblasts but clearly detectable in the mature osteoblasts of the secondary spongiosa. When we compared expression of osteocalcin and collagen type 1 mRNA in mature cuboidal and older flat osteoblasts, we found that the amount of osteocalcin mRNA relative to collagen type 1 mRNA was higher in flat osteoblasts than in cuboidal osteoblasts. In odontoblasts, however, the steady state level of collagen type 1 mRNA was higher in the older coronal odontoblasts, and the level of osteocalcin message lower, when compared to the younger apical odontoblasts. The results indicate that relative levels of osteocalcin and collagen mRNA in osteoblasts and odontoblasts vary depending on the stage in their secretory lifetime. This heterogeneity of the osteoblast and odontoblast population suggests that the composition of the matrix produced by these cells also differs.