Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice

Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of i nfantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt ), heterozygous and homozygous TNAP null mice. In vitro release of Ca-45 fr om calvarial bones, with and without stimulation with parathyroid hormone ( PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAPS+/-, and TNAP- /- calvarial osteoblasts revealed no differences in the rate of protein syn thesis or in the expression levels of messenger RNAs (mRNAs) for osteoponti n (OP), osteocalcin (OC), collagen type I, core binding factor alpha 1 (Cbf a 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH , tumor necrosis factor alpha (TNF-alpha) or IL-1 beta was similar in all g enotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteobla sts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Minerali zation also was delayed in TNAP+/- osteoblast cultures compared with cultur es of wt osteoblasts. Incubation with media supplemented with recombinant T NAP, bur not with enzymatically inactive TNAP, restored mineralization in k o osteoblast cultures. Our data provide evidence that osteoblasts in TNAP n ull mice differentiate normally but are unable to initiate mineralization i n vitro. The fact that even heterozygous osteoblasts show delayed mineraliz ation provides a rationale for the presence of bone disease in carriers of hypophosphatasia.