ERK1/2-activated de Novo Mapkapk2 Synthesis Is Essential for Osteogenic Growth Peptide Mitogenic Signaling in Osteoblastic Cells

Symone M San Miguel,Malka Namdar-Attar,Tommy Noh,Baruch Frenkel,Itai Bab

doi:10.1074/jbc.m503861200

Abstract

In osteoblasts, the mitogen-activated protein kinases ERK1/2 and p38 as well as the cAMP-response element-binding protein (CREB) have been implicated in the regulation of proliferation and differentiation. The osteogenic growth peptide (OGP) is a 14-mer bone cell mitogen that increases bone formation and trabecular bone density and stimulates fracture healing. OGP-(10-14) is the physiologically active form of OGP. Using gene array analysis, real-time reverse transcription-PCR, and immunoblot and DNA synthesis assays we show here that in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures the OGP-(10-14) mitogenic signaling is critically dependent on de novo synthesis of mitogen-activated protein kinase-activated protein kinase 2 (Mapkapk2) mRNA and protein. The increase in Mapkapk2 occurs following short term (5-60 min) stimulation of ERK1/2 activity by OGP-(10-14); phosphorylation of p38 remains unaffected. Downstream of Mapkapk2, CREB is phosphorylated on Ser(133) leading to its enhanced transcriptional activity. That these events are critical for the OGP-(10-14) mitogenic signaling is demonstrated by blocking the effects of OGP-(10-14) on the ERK1/2 pathway, Mapkapk2, CREB, and DNA synthesis using the MEK inhibitor PD098059. The OGP-(10-14) stimulation of CREB transcriptional activity and DNA synthesis is also blocked by Mapkapk2 siRNA. These data define a novel mitogenic signaling pathway in osteoblasts whereby ERK1/2 stimulation of CREB phosphorylation and transcriptional activity as well as DNA synthesis are critically dependent on de novo Mapkapk2 synthesis.

Highlights

Coupled receptors [1, 2]
osteogenic growth peptide (OGP)-(10 –14) Mitogenic Signaling in Osteoblasts Involves Activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) but Not p38—Previously, we showed by immunoprecipitation and kinase assays that treatment of osteoblastic cells with OGP
In this study we show that the OGP-(10 –14) mitogenic signaling in osteoblastic cells involves rapid phosphorylation of ERK1/2 and de novo Mapkapk2 mRNA and protein synthesis

Summary

EXPERIMENTAL PROCEDURES

Materials—OGP-(10 –14), amino acid sequence Tyr-Gly-Phe-GlyGly, molecular mass 499.7 kDa, was supplied by Polypeptides Laboratories, Inc. (Torrance, CA) and provided by Abiogen Pharma Spa (Pisa, Italy). Thereafter, the cells were incubated for various time periods, ranging from 5 min to 8 h, in the same medium with or without OGP-(10 –14) and MAP kinase inhibitors. After 5 and 30 h of incubation in control and Mapkapk siRNA, respectively, the cells were serum-starved for 2 h and challenged with OGP-(10 –14). RNA Isolation—Total RNA was isolated from MC3T3 E1 and NeMCO cells incubated for 4 or 8 h with or without OGP-(10 –14) and MAP kinase inhibitors using TRI reagent kit (Molecular Research Center, Inc., Cincinnati, OH) followed by a phenol-chloroform phase extraction and isopropyl precipitation. CDNA Macroarray Analysis—RNA samples displaying an A260/280 ratio of 1.8 or higher and well defined 18 S and 28 S ethidium bromidestained bands were subjected to expression analysis using mouse MAP kinase signaling pathways gene array kit The Mann-Whitney U test was used for the cDNA macroarray analysis

TABLE ONE

Expression levela

DISCUSSION