Runx2 Regulates G Protein-coupled Signaling Pathways to Control Growth of Osteoblast Progenitors

Nadiya M Teplyuk,Mario Galindo,Viktor I Teplyuk,Jitesh Pratap,Daniel W Young,David Lapointe,Amjad Javed,Janet L Stein,Jane B Lian,Gary S Stein,Andre J Van Wijnen

doi:10.1074/jbc.m802453200

Abstract

Runt-related transcription factor 2 (Runx2) controls lineage commitment, proliferation, and anabolic functions of osteoblasts as the subnuclear effector of multiple signaling axes (e.g. transforming growth factor-beta/BMP-SMAD, SRC/YES-YAP, and GROUCHO/TLE). Runx2 levels oscillate during the osteoblast cell cycle with maximal levels in G(1). Here we examined what functions and target genes of Runx2 control osteoblast growth. Forced expression of wild type Runx2 suppresses growth of Runx2(-/-) osteoprogenitors. Point mutants defective for binding to WW domain or SMAD proteins or the nuclear matrix retain this growth regulatory ability. Hence, key signaling pathways are dispensable for growth control by Runx2. However, mutants defective for DNA binding or C-terminal gene repression/activation functions do not block proliferation. Target gene analysis by Affymetrix expression profiling shows that the C terminus of Runx2 regulates genes involved in G protein-coupled receptor signaling (e.g. Rgs2, Rgs4, Rgs5, Rgs16, Gpr23, Gpr30, Gpr54, Gpr64, and Gna13). We further examined the function of two genes linked to cAMP signaling as follows: Gpr30 that is stimulated and Rgs2 that is down-regulated by Runx2. RNA interference of Gpr30 and forced expression of Rgs2 in each case inhibit osteoblast proliferation. Notwithstanding its growth-suppressive potential, our results surprisingly indicate that Runx2 may sensitize cAMP-related G protein-coupled receptor signaling by activating Gpr30 and repressing Rgs2 gene expression in osteoblasts to increase responsiveness to mitogenic signals.

Highlights

Transcription factor 2) is a critical regulator of bone development by driving osteoblast differentiation and formation of a bone-specific mineralized extracellular matrix [1,2,3,4]
Domains—We have previously shown that Runx2 deficiency increases the proliferative potential of osteoprogenitors and that re-introduction of wild type Runx2 into primary calvaria cells restores stringent cell growth control [5]
We have characterized mechanisms that account for the ability of Runx2 to control osteoblast proliferation using an integrated strategy in which we investigated, first, what functions of Runx2 are required for cell growth control and, second, what downstream targets mediate its growth regulatory function

Summary

The abbreviations used are

TGF␤, transforming growth factor-␤; GPCR, G protein-coupled receptor; PTH, parathyroid hormone; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; qRT, quantitative reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IRES, internal ribosome entry site; GFP, green fluorescent protein; EGFP, enhanced GFP; ␣-MEM, minimum Eagle’s ␣-medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; siRNA, short interfering RNA; FACS, fluorescence-activated cell sorting; MOPS, 4-morpholinepropanesulfonic acid; PMA, phorbol myristate acetate; CREB, cAMP-responsive elementbinding protein; BMP, bone morphogenetic protein; Cdk, cyclin-dependent kinase. Runx Control of G protein Signaling cohort of cofactors (Ͼ24 partner proteins) that support activation or repression of Runx target genes in osteoblasts Runx transduces TGF␤/BMP2 signaling through direct interactions with SMAD proteins that bind to a composite protein domain (“NMTS/SMID”) that mediates transcriptional activation and subnuclear targeting [31, 38]. Despite the growing number of Runx partner proteins that modify or modulate its transcriptional activity, there is limited understanding of the signaling pathways and Runx cofactors that control the growth regulatory potential of Runx and the downstream expression programs that mediate its cellular functions. Our studies revealed that DNA binding and C-terminal transcriptional functions of Runx are essential for cell growth control. Our data suggest that Runx may regulate G protein-signaling pathways to modify how osteoblasts respond to mitogenic stimuli

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION