RETINOIC ACID-INDUCED MAFB EXPRESSION DURING MYELOID CELL DIFFERENTIATION IN CULTURE AND BONE CHONDROGENESIS IN VIVO

Open Access
- Author:
- Zhang, Yao
- Graduate Program:
- Nutrition
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 17, 2011
- Committee Members:
- A. Catharine Ross, Committee Chair/Co-Chair
Penny Kris-Etherton, Committee Member
Okhee Han, Committee Member
Andrea M. Mastro, Committee Member - Keywords:
- chondrocytes
bone
THP-1 cells
transcription factor
retinoic acid
differentiation - Abstract:
- Vitamin A and its active metabolite, all-trans retinoic acid (RA) regulate many physiological processes, such as skeletal development, cell differentiation, and immune functions. RA has been shown to induce monocytic cell differentiation and enhance phagocytic activity. Moreover, RA concentration is higher in hypertrophic chondrocytes, play an essential role in inducing chondrocyte terminal differentiation. MafB, a basic leucine-zipper transcription factor, is involved in development and monocyte-macrophage differentiation. Microarray analysis conducted by our laboratory revealed that MafB was one of the most strongly regulated genes by RA in THP-1 human monocytic cells. Therefore, we have hypothesized that RA-induced MafB is a mediator in the differentiation of monocytic cells and chondrocytes. To address this hypothesis, my dissertation was divided into two parts. The first part was to investigate the role of MafB in RA-induced monocytic cells differentiation using THP-1 cells as a model. There were three aims under this part: 1) to clarify MafB expression under the condition of RA treatment in monocytic THP-1 cells; 2) to determine the downstream genes of MafB during RA- and tumor necrosis factor (TNF) α-induced monocytic differentiation; 3) to determine the requirement of MafB in the RA- and TNFα-induced monocytic differentiation. The second part of my dissertation was to clarify the roles of MafB in RA-induced chondrogenesis using rats as an animal model. Two aims was developed: 1) to investigate the alteration of MafB expression and other bone makers during the rat limb development under different maternal vitamin A status and oral vitamin A and RA supplementation; 2) to determine MafB expression and function during the RA-induced chondrocyte terminal differentiation. In THP-1 human monocytic cells, MafB mRNA and protein levels were up-regulated by RA in a dose and time-dependent manner while, additionally, RA and TNFα synergistically regulated MafB expression, mainly at the transcriptional level. SPOCK1, Blimp1 and CCL2 were indentified as target genes of MafB. Binding activity of MafB to the promoters of each of the target genes was increased in the RA- and TNFα- treated cells. Conversely, reducing MafB protein by microRNA silencing significantly decreased the expression of SPOCK1, Blimp1 and CCL2. Moreover, the reduction in MafB resulted in decreased cell differentiation and phagocytic activity. Together, RA- and TNFα-induced MafB may play a role in monocytic cell differentiation through transcriptional regulation of Blimp1, CCL2 and SPOCK1. In a rat study, maternal dietary vitamin A (VA) intake was represented the range of VA in most human diet: VA marginal, VA adequate, and VA supplemented. Their offspring were studied at birth (P0) and postnatal day 7 (P7). Half of the newborns received an oral supplement of VARA, or oil placebo. Maternal VA did not affect bone mineralization assessed by von Kossa staining; however, significantly differences (P<0.01) in the lengths of pups’ femur and tibia were observed across different maternal VA diets. Only in the VA-marginal group, oral feeding with VARA significantly increased the length of the hypertrophic zone, close to the length in pups from VA-adequate group. Neither type X nor type II collagen mRNA was altered by maternal VA status, although VARA intake significantly enhanced type X collagen mRNA levels. However, one of the most important finding was that VARA-treated pup femurs from VA-supplemented dams decreased aggrecan, a major component of cartilage matrix, and increased matrix metalloproteinase (MMP)13 expression, which catalyzes degradations of aggrecan and collagens. This result suggests that maternal VA supplementation plus neonatal VARA treatment could potentially be unfavorable for early bone development. Moreover, MafB was localized in both proliferative and hypertrophic chondrocytes in the growth plate. The expression in hypertrophic zone was stronger when maternal VA intake was higher. In cultured chondrocytes, RA dose-dependently increased MafB expression. MafB knockdown in chondrocytes resulted in an increase of aggrecan and a decrease of RA-induced MMP13. These results implicate RA-induced MafB as a regulator of chondrocyte gene expression and matrix formation via the control of aggrecan and MMP13 expression. In conclusion, my study have provided a strong evidence that MafB was induced by RA, mediating RA-induced cell differentiation in both monocytic cells and primary chondrocytes.