Stathmin levels in growth plate chondrocytes are modulated by vitamin D-3 metabolites and transforming growth factor-beta 1 and are associated with proliferation

Stathmin is a highly conserved, phosphorylated cytosolic protein that is fo und at decreased levels in all cells as they become more terminally differe ntiated, or when they decrease in their rate of proliferation. This study e xamined the hypothesis that stathmin levels in growth plate chondrocytes de creases as endochondral maturation increases. To test this hypothesis, we u sed a costochondral growth plate chondrocyte cell culture model. Cells deri ved from the resting zone (RC) express twice as much stathmin mRNA in cultu re and have twice as much stathmin protein as cells derived from the post p roliferative growth zone ([GC]; prehypertrophic and upper hypertrophic cell zones). Stathmin levels in vivo were assessed by immunohistochemistry. To assess the effects of agents that modulate proliferation and differentiatio n, RC and GC chondrocytes were cultured in the presence of 10(-10) to 10(-8 ) M1 alpha ,25-(OH)(2)D-3, which regulates proliferation in both cell types but affects differentiation of only GC cells, or 10(-9) to 10(-7) M 24R,25 -(OH)(2)D-3, which regulates differentiation and maturation of RC cells but decreases proliferation of GC cells. In addition, RC cells were treated wi th 0.44 or 0.88 ng/mL of recombinant human transforming growth factor beta1 (rhTGF-beta1), which stimulates proliferation of RC cells and regulates pr oteoglycan production, but not alkaline phosphatase activity. Stathmin prot ein levels were determined using quantitative immunoblots, with recombinant human stathmin as a standard. The results show that stathmin levels are as sociated with proliferation. Proliferating chondrocytes in vivo exhibited h igher levels of immunoreactive stathmin than either RC or GC cells in the g rowth plate. In culture, 1 alpha ,25-(OH)(2)D-3 caused a dose-dependent dec rease in stathmin in RC and GC cells within 24 h. 24R, 25-(OH)(2)D-3 also r educed stathmin levels in GC cells within 24 h but only affected RC cells a fter prolonged exposures (96 h), at which time RC cells express a GC-like p henotype. rhTGF-beta1 caused an increase in stathmin levels in RC cells. St athmin levels are sensitive to protein kinase C (PKC) in other cells. Inhib ition of PKC with chelerythrine had no effect on the response of RC cells t o 1 alpha ,25-(OH)(2)D-3 but it blocked the effect of rhTGF-beta1, indicati ng that decreases in stathmin by vitamin D-3 metabolites may not be modulat ed by PKC, whereas increases in stathmin via rhTGF-beta1 may be regulated v ia a PKC-dependent mechanism. These results support the hypothesis that con stitutively expressed levels of stathmin are related to cell maturation sta te and that they are modulated by factors that regulate proliferation.