Betacellulin inhibits osteogenic differentiation a

Authors: Damian C Genetos Rameshwar R Rao Martin A Vidal

Publish Date: 2010/02/18

Volume: 340, Issue: 1, Pages: 81-89

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Abstract

Cellular signaling via epidermal growth factor EGF and EGFlike ligands can determine cell fate and behavior Osteoblasts which are responsible for forming and mineralizing osteoid express EGF receptors and alter rates of proliferation and differentiation in response to EGF receptor activation Transgenic mice overexpressing the EGFlike ligand betacellulin BTC exhibit increased cortical bone deposition however because the transgene is ubiquitously expressed in these mice the identity of cells affected by BTC and responsible for increased cortical bone thickness remains unknown We have therefore examined the influence of BTC upon mesenchymal stem cell MSC and preosteoblast differentiation and proliferation BTC decreases the expression of osteogenic markers in both MSCs and preosteoblasts interestingly increases in proliferation require hypoxiainducible factoralpha HIFα as an HIF antagonist prevents BTCdriven proliferation Both MSCs and preosteoblasts express EGF receptors ErbB1 ErbB2 and ErbB3 with no change in expression under osteogenic differentiation These are the first data that demonstrate an influence of BTC upon MSCs and the first to implicate HIFα in BTCmediated proliferationThe skeleton continually undergoes alterations in size and shape in response to humoral mechanical and paracrine stimuli Degradation of preexisting bone by osteoclasts and ensuing replacement with osteoid by osteoblasts require concerted activity in order to maintain skeletal homeostasis and to prevent pathologic conditions such as osteoporosis or osteopetrosis Osteoblast number is partially governed by the proliferation and osteogenic differentiation of mesenchymal stem cell MSC precursors hence stimuli that enhance MSC number or osteogenic differentiation emerge as ideal methods to combat bone loss resulting from pharmacology or agingEpidermal growth factor EGF mediates diverse processes of proliferation differentiation adhesion and survival Citri and Yarden 2006 Yarden and Sliwkowski 2001 The EGF family of ligands consists of EGF amphiregulin betacellulin BTC epiregulin epigen heparinbinding EGFlike growth factor HBEGF and transforming growth factoralpha TGFα Harris et al 2003 Xian 2007 EGF signaling occurs through ErbB receptor tyrosine kinases of which ErbB1/EGFR is bound by all seven family members Three members EGF amphiregulin and TGFα can only bind to ErbB1 whereas BTC epiregulin and HBEGF can also bind to ErbB4 to form ErbB1/ErbB4 heterodimers ErbB2 although lacking any known cognate ligand is the preferred coreceptor for ErbB1 and the heterodimerization of ErbB2 and ErbB1 generates more potent signaling than ErbB1 homodimers Qin et al 2005 Yarden 2001 Yarden and Sliwkowski 2001 ErbB3 is a pseudokinase and therefore lacks any traditional catalytic kinase domain Once bound by EGF or EGFlike ligand homo or heterodimerized ErbB receptors initiate multiple signaling pathways that affect proliferation survival and differentiation and that are mediated RasRafmitogenactivated protein kinase RasRafMAPK Yarden and Sliwkowski 2001 the phosphatidylinositol3kinase/Akt pathway Xian 2007 and phospholipase Cγ/protein kinase C Yarden and Sliwkowski 2001EGF and related ligands exert disparate effects upon osteoblast differentiation and proliferation In vitro studies have revealed that EGF inhibits bone nodule formation Antosz et al 1987 and decreases alkaline phosphatase ALP activity Nicolas et al 1990 Similar findings have been reported for osteoblastic cells treated with amphiregulin Qin et al 2005 Further overexpression of ErbB1 in ROS rat osteosarcoma cells decreases the expression of osteoblastic markers Chien et al 2000 Comparable results are observed with in vivo models EGF transgenic mice under the control of a βactin promoter demonstrate a 20–50 reduction in body weight Wong et al 2000 and decreased cortical bone thickness compared with wildtype controls Chan and Wong 2000 Similar observations including impaired longitudinal growth and overall disproportionate skeletal growth are detected in mice transgenic for BTC Schneider et al 2005 Whereas EGF suppresses the osteoblastic phenotype it demonstrates mitogenic effects upon osteoblastic cells Loza et al 1995 Nicolas et al 1990 VandePol et al 1989 as do amphiregulin and TGFα Qin et al 2005 Thus a general theme regarding EGF signaling in bone seems to be increased proliferation and decreased osteogenic differentiation Schneider et al 2009 have recently demonstrated that BTC transgenic mice have enhanced cortical but reduced trabecular bone thickness To date however no study has examined the influence of BTC upon osteoprogenitor proliferation or differentiation We report that BTC attenuates the osteogenic differentiation of MSC and committed preosteoblastic cells whilst simultaneously increasing their proliferation through a mechanism involving the stabilization of hypoxiainducible factor HIF These are the first data to demonstrate an influence of BTC upon MSC and the first to implicate HIF in BTCmediated proliferationHuman MSCs hMSCs were purchased from Lonza cells were isolated from iliac crest bone biopsies from a 19yearold male AfricanAmerican a 20yearold male AfricanAmerican and an 18yearold male Caucasian with no known pathologic conditions hMSCs passages 2–6 were cultured in αMEM supplemented with 10 fetal bovine serum FBS Invitrogen and 1 penicillin/streptomycin P/S Invitrogen and maintained in a humidified incubator at 37°C with 5 CO2 MC3T3E1 clone 14 preosteoblastic cells were purchased from American Type Culture Collection and cultured in αMEM supplemented with 10 FBS and 1 P/S and maintained in a humidified incubator at 37°C with 5 CO2 Cells were subcultured with 005 trypsin/EDTA when 80 confluenthMSCs or MC3T3E1 cells were seeded into 6well plates at a density of 3000 cells/cm2 When 70 confluent media was replaced with osteogenic differentiation media consisting of 10 FBS 1 P/S 5 mM βglycerophosphate acid VWR International and 50 µg/ml Lascorbic acid 2phosphate Waco in αMEM InvitrogenTotal RNA was collected with RNeasy Mini kit Qiagen according to the manufacturer’s instructions and including oncolumn digestion of genomic DNA RNA quality was assessed by the ratio of absorbance at 260 nm and 280 nm Total RNA 02–1 μg was reversetranscribed with a Superscript FirstStrand Synthesis System Invitrogen Quantitative reverse transcription with the polymerase chain reaction RTPCR was performed by using the TaqMan Universal PCR Master Mix Applied Biosystems on a Mastercycler realplex2 Eppendorf proprietary primers and probes whose sequences are not disclosed were purchased from Applied Biosystems Amplification conditions were 50°C for 2 min 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 30 s Quantitative PCR results were first normalized to ribosomal protein 13 Rpl13 transcript level to yield ΔCt Results are expressed as 2 Delta rmCt or 2 Delta Delta rmCt after Livak and Schmittgen 2001

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