Abstract
Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.
Highlights
The mechanisms by which Hivep3 regulates the osteochondrogenesis remain elusive
We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum
We initially hypothesized that Hivep3 may inhibit bone morphogenetic protein (BMP) signaling to suppress osteoblast differentiation; the expression of the direct target genes of the BMPSmad pathway, Id1 or Smad6, was not altered by Hivep3 knockdown in osteoblasts
Summary
The mechanisms by which Hivep regulates the osteochondrogenesis remain elusive. Results: Knockdown of Hivep down-regulated Alg expression. Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. A D-domain motif within Hivep mediates the interaction with and inhibition of ERK mitogen-activated protein kinase (MAPK), thereby inhibiting Wnt/Lrp signaling through regulation of the activity of a downstream mediator glycogen synthase kinase 3- (Gsk3) This interaction results in the suppression of subsequent osteoblast differentiation [13]. In chondrogenesis of ATDC5 cells, Hivep induced the expression of cAMP-responsive element binding-protein 3-like 2 (Creb3l2), an endoplasmic reticulum (ER) stress transducer crucial for chondrogenesis [18], suggesting a possible role for Hivep in physiological mild ER stress. This study is the first to show a linkage between an asparagine-linked glycosylation mannosyltransferase gene and osteochondrogenesis
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