Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long-term subculturing. In order to study in vitro age-related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast-specific genes in early-passage (< 50% lifespan completed) with those of late-passage cells (> 90% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late-passage cells compared to early-passage cells, suggesting an in vitro age-related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic cells inhibited their proliferation by 57 +/- 1% and stimulated steady state mRNA levels of AP (1.7 +/- 0.1-fold) and OC (1.8 +/- 0.2-fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7 +/- 0.2-fold) and OC (3.0 +/- 0.3-fold) in late-passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age-related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment.