Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.
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 Ca2+ 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 Ca2+ channel function.


BACKGROUND
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.


METHODS
Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 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 alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ 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 Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization.


CONCLUSIONS
These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.
Zahanich
Ihor
I
Institut für Pharmakologie und Toxikologie, Medizinische Fakultät Carl Gustav Carus der TU Dresden, Dresden, Germany.
Graf
Eva M
EM
Heubach
Jürgen F
JF
Hempel
Ute
U
Boxberger
Sabine
S
Ravens
Ursula
U
D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000096:biosynthesis* / Q000235:genetics*; D020747:Calcium Channels, T-Type / Q000096:biosynthesis* / Q000235:genetics*; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D003470:Culture Media / Q000378:metabolism / Q000494:pharmacology; D017931:DNA Primers / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D005434:Flow Cytometry; D005994:Glycerophosphates / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D053495:Osteopontin; D053244:Osteoprotegerin; D018408:Patch-Clamp Techniques; D010710:Phosphates / Q000737:chemistry; D016133:Polymerase Chain Reaction; D053245:RANK Ligand; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000096:biosynthesis
C079146:CACNA1C protein, human; C121855:CACNA1G protein, human; D020746:Calcium Channels, L-Type; D020747:Calcium Channels, T-Type; D002352:Carrier Proteins; D003470:Culture Media; D017931:DNA Primers; D005994:Glycerophosphates; D006023:Glycoproteins; C419385:L-type calcium channel alpha(1C); D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D053244:Osteoprotegerin; D010710:Phosphates; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; C505686:TNFRSF11A protein, human; C506030:TNFRSF11B protein, human; C505688:TNFSF11 protein, human; C506032:Tnfrsf11b protein, rat; D053495:Osteopontin; D012313:RNA; D003907:Dexamethasone; D000469:Alkaline Phosphatase; D009543:Nifedipine; D001205:Ascorbic Acid; D002118:Calcium; C031463:beta-glycerophosphoric acid