Negative regulation of osteoblast differentiation by cellular retinoic acid binding protein-II (CRABP-II)

Abstract

Cellular retinoic acid binding protein (CRABP) is a family of intracellular lipid-binding proteins (iLBPs). CRABP-II is known to bind retinoic acid (RA) in the cytoplasm and transport RA and RAR complex into the nucleus for gene transcription. In bone metabolism, there are two main types of cells called osteoblasts and osteoclasts. Osteoblast is the bone forming cell while osteoclast is a bone resorbing cell. Vitamin A is known to negatively affect osteoblasts. One of the study reported that high dose of vitamin A increases bone fracture in both animal and human. However, according to another study, the active form of vitamin A forms a complex with RA receptor (RAR) and stimulates the differentiation of ostoblastogenesis from mesenchymal cells. In addition, the level of CRABP-II was shown to increase as osteoblast differentiates in osteoblast gene array study. The purpose of this study was to verify the exact role of CRABP-II in osteoblastogenesis. For the study, primary mouse calvarial osteoblast was mainly used along with the MC3T3-E1 cell. The level of CRABP- II was measured by RTPCR and western blot. The effect of knockdown and overexpression of CRABP-II in osteoblasts was analyzed by ALP staining and Alizarin Red S staining. To see the proliferation of the overexpressed CRABP-II in osteoblasts, CCK and BrdU were used. The mRNA level and the protein expression of CRABP-II was observed in both primary osteoblasts and MC3T3-E1 cells as the cells become differentiated. However, CRABP-II was not expressed in osteoclasts. Knockdown of CRABP-II in both osteoblasts and MC3TC-E1 cells resulted in an increase in the intensity of ALP staining. A significant increase of mineralization was detected in CRABP-II knockdown cells. To determine the effect of CRABP-II overexpression in primary osteoblasts, mouse calvarial osteoblasts were transduced with a retroviral system. CRABP-II overexpressing cells had larger mineralized area that was stained with Alizarin Red S. Both BrdU and CCK assays presented similar results of no difference between the possible negative regulation by CRABP-II of osteoblastogenesis.