Establishment and hormonal regulation of a conditionally transformed preosteocytic cell line from adult human bone

Abstract

Osteocytes are differentiated forms of osteoblasts that arise upon entrapment within the bone matrix. In this report, we describe the establishment and hormonal regulation of the first conditionally transformed human preosteocytic cell line. Primary adult bone cells were obtained from protease digestion of cancellous chips. The cells were infected with adenovirus-ori SV40 tsA 209, which encodes for a temperature-sensitive large T-antigen. After immortalization, we isolated a clone designated HOB-01-C1. This cell line expressed the mutant T-antigen and proliferated at the permissive temperature (34 C) but stopped dividing at the nonpermissive temperature (39-40 C). Electron microscopy of cells incubated at 39 C demonstrated the presence of preosteocytic cellular processes, some of which appeared to form gap junctions or were rich in microfilaments. The clone expressed α1 type (I) procollagen messenger RNA (mRNA) and secreted type I procollagen C peptide at both temperatures, and this expression was elevated 1.6-fold to 1.8-fold at 40 C. The cells expressed very low basal levels of alkaline phosphatase activity (~0.02 nmol/min · mg), which was increased 2- to 5-fold in a dose-dependent manner by 0.1-100 nM 1α,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures. Vitamin D3 also increased osteocalcin secretion in a dose-dependent manner when the clone was maintained at 34 C (~6-fold), and this stimulation was enhanced >5-fold at 40 C. In contrast to the low expression of alkaline phosphatase, the cells secreted high amounts of osteocalcin in response to vitamin D3 (~15 ng/mg cell protein); this biochemical profile also resembled that of preosteocytes. Alizarin red-S histochemical staining demonstrated that these cells rapidly produced mineralized nodules at both temperatures. PTH (10 and 100 nM) had no effect on the intracellular accumulation of cAMP at 34 C but stimulated a 14- to 18-fold increase in the production of this second messenger at 40 C. In contrast, 100 nM prostaglandin E2 and 1 μM forskolin stimulated cAMP synthesis better at 34 C. Western blot analysis indicated that the cells expressed CD44, a putative osteocytic marker, at both temperatures. Finally, interleukin-1β and tumor necrosis factor-α (1-1000 pM) stimulated dose- dependent increases in the secretion of interleukin-6 and monocyte chemoattractant protein-1 at 34 C and 40 C. We conclude that the HOB-01-C1 cell line has a preosteocytic phenotype. Moreover, these cells respond to calcitropic hormones and bone resorbing cytokines.