Calbindin-D28K and -D9K and 1,25(OH)2 vitamin D3 receptor immunolocalization and mineralization induction in long-term primary cultures of rat epiphyseal chondrocytes.
Rat epiphyseal plat chondrocytes were grown on glass slides, as nonadhering monolayer cultures for up to 6 weeks. Chondrocyte growth, differentiation and maturation, matrix formation and mineralization, and the temporospatial distribution of the vitamin D-dependent calcium-binding proteins, calbindin-D9K and -D28K, and the 1,25(OH)2D3 receptor (VDR), were all monitored. Chondrocytes became confluent in 2.5 weeks, differentiated to acquire a chondrocyte (polygonal) morphology, produced extracellular matrix, and finally formed a true monolayer mineralizing cartilaginous tissue, with all the stages of chondrocyte development within a single culture. beta-Glycerophosphate promoted initial matrix mineralization in 4 weeks and accelerated cell differentiation. High nominal calcium and ascorbic acid were needed for abundant matrix formation. VDR occurred at all differentiation stages, in the nuclei and nucleoli and in the cytoplasm. Calbindin-D28K and -D9K were not coexpressed. Calbindin-D28K was found in prechondroblasts, chondroblasts, and in newly differentiated chondrocytes. It was cytoplasmic in prechondroblasts and subsequently also in the perinuclear region and in nuclei, suggesting migration to the nuclear chromatin. Calbindin-D28K was nuclear only in newly differentiated chondrocytes in vitro and was not found in mature chondrocytes. In contrast, calbindin-D9K was present in the cytoplasm of mature and hypertrophic chondrocytes only. It was first in the cell body and eventually migrated within and to the far end of long cell processes with a decreasing cytoplasmic concentration showed by decreased immunostaining intensity, and ultimately hypertrophy of chondrocytes in culture. These in vitro patterns of calbindins-D and VDR accurately reflect their in vivo distributions. The genomic action of vitamin D, in vitro, resulted in the synthesis of nuclear VDR and calbindins-D.(ABSTRACT TRUNCATED AT 250 WORDS)
Balmain
N
N
INSERM U120, alliée C.N.R.S., Hôpital R. Debré, Paris, France.
von Eichel
B
B
Toury
R
R
Belquasmi
F
F
Hauchecorne
M
M
Klaus
G
G
Mehls
O
O
Ritz
E
E
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D015519:Bone Density / Q000502:physiology*; D064092:Calbindin 1; D064026:Calbindins; D002113:Calcification, Physiologic / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004396:Coloring Agents; D005109:Extracellular Matrix / Q000378:metabolism; D005994:Glycerophosphates / Q000494:pharmacology; D006132:Growth Plate / Q000166:cytology* / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D018167:Receptors, Calcitriol / Q000378:metabolism*; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*
C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D004396:Coloring Agents; D005994:Glycerophosphates; D009419:Nerve Tissue Proteins; D018167:Receptors, Calcitriol; D064030:S100 Calcium Binding Protein G; C576508:S100g protein, rat; D001205:Ascorbic Acid; D002118:Calcium; C031463:beta-glycerophosphoric acid