The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts

Abstract

Heterogeneous nuclear ribonucleoprotein (hnRNP) C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D (1,25(OH)₂D) by functioning as a vitamin D response element-binding protein (VDRE-BP). hnRNPC acts a tetramer of hnRNPC1 (huC1) and hnRNPC2 (huC2), and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25(OH)₂D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25(OH)₂D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a “self-cleaving” 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25(OH)₂D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.

Vitamin D: Species-specific effects of heterogeneous ribonucleoproteins in mice

Species-specific assembly of RNA-binding proteins determines the effects of vitamin D in osteoblasts (bone-building cells). Previous studies revealed that overexpression of human heterogeneous ribonucleoprotein (hnRNP) isoforms C1 and C2 together, or individually, in human osteoblasts inhibits vitamin D-induced gene expression, which is associated with vitamin D-resistant bone disease. Martin Hewison and John S. Adams at the University of California, Los Angeles, and colleagues recently discovered that overexpression of each human hnRNPC isoform individually in mouse osteoblasts was insufficient to recapitulate the vitamin D-resistant phenotype. To exert their effects, hnRNPC proteins form tetramers and this study identified a key amino acid in human hnRNPCs (asparagine 200) required for formation of functional tetramers. However, mouse hnRNPCs contain a serine at this position that prevents stabilization of inter-species tetramers, suggesting that species-specific tetramerization of hnRNPC is crucial for vitamin D-dependent gene expression in mouse osteoblasts.

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