Premium
Molecular and Functional Expression of Voltage‐Operated Calcium Channels During Osteogenic Differentiation of Human Mesenchymal Stem Cells
Author(s) -
Zahanich Ihor,
Graf Eva M,
Heubach Jürgen F,
Hempel Ute,
Boxberger Sabine,
Ravens Ursula
Publication year - 2005
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.050521
Subject(s) - alkaline phosphatase , mesenchymal stem cell , chemistry , microbiology and biotechnology , cellular differentiation , osteopontin , voltage dependent calcium channel , osteoblast , calcium , calcium channel , l type calcium channel , runx2 , endocrinology , biochemistry , biology , in vitro , enzyme , gene , organic chemistry
We used the patch‐clamp technique and RT‐PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L‐type Ca 2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L‐type Ca 2+ channel function. Introduction : During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage‐operated calcium channels (VOCCs), including L‐type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. Materials and Methods : Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, β‐glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca 2+ channel α1 subunits was shown by semiquantitative or single cell RT‐PCR. Voltage‐activated calcium currents of hMSCs were measured with the whole cell voltage‐clamp technique. Results : mRNA for the pore‐forming α1C and α1G subunits of the L‐type and T‐type Ca 2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L‐type Ca 2+ currents to a subpopulation of hMSCs was confirmed by single cell RT‐PCR, where mRNA for the α1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine‐sensitive L‐type Ca 2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L‐type Ca 2+ currents were significantly larger than cells without Ca 2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L‐type Ca 2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. Conclusion : These results suggest that, in the majority of hMSCs, Ca 2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L‐type Ca 2+ channels does not affect early osteogenic differentiation of hMSCs.