Beta-arrestin2 regulates parathyroid hormone effects on a p38 MAPK and NFkappaB gene expression network in osteoblasts

Abstract

Interaction of the cytoplasmic adaptor molecule beta-arrestin2 with the activated parathyroid hormone (PTH)/PTHrP receptor inhibits G protein mediated signaling and triggers MAPKs signaling. In turn, the effects of both intermittent (i.) and continuous (c.) PTH on bone are altered in beta-arrestin2-deficient (Arrb2(-/-)) mice. To elucidate the expression profile of bone genes responsive to PTH and targeted for regulation by beta-arrestin2, we performed microarray analysis using total RNA from primary osteoblastic cells isolated from wild-type (WT) and Arrb2(-/-) mice. By comparing gene expression profiles in cells exposed to i.PTH, c.PTH or vehicle (Veh) for 2 weeks, we found that i.PTH specifically up-regulated 215 sequences (including beta-arrestin2) and down-regulated 200 sequences in WT cells, about two-thirds of them being under the control of beta-arrestin2. In addition, beta-arrestin2 appeared necessary to the down-regulation of a genomic cluster coding for small leucin-rich proteins (SLRPs) including osteoglycin, osteomodulin and asporin. Pathway analyses identified a main gene network centered on p38 MAPK and NFkappaB that requires beta-arrestin2 for up- or down-regulation by i.PTH, and a smaller network of PTH-regulated genes centered on TGFB1, that is normally repressed by beta-arrestin2. In contrast the expression of some known PTH gene targets regulated by the cAMP/PKA pathway was not affected by the presence or absence of beta-arrestin2 in osteoblasts. These results indicate that beta-arrestin2 targets prominently p38 MAPK- and NFkappaB-dependent expression in osteoblasts exposed to i.PTH, and delineates new molecular mechanisms to explain the anabolic and catabolic effects of PTH on bone.

Fig.1. Osteoblast differentiation and gene expression in response to PTH

(A) Effects of intermittent PTH (i.PTH), continuous PTH (c.PTH) or vehicle (Veh) on mineralization of primary osteoblasts isolated from WT and Arrb2^−/− mice. Mineralization was revealed by Alizarin Red staining. (B) Scheme representing the step-by-step approach used to delineate the sequences/genes regulated by i.PTH and targeted for regulation by β-arrestin2. See Materials and methods section for details.

Fig. 2. Relevant canonical pathways associated with the 415 and the 39 sequences regulated uniquely by i.PTH (A) and c.PTH (B) in WT osteoblasts

The threshold line across the bars represents the point where for a given pathway the significance value for the observation is of 0.05. The ratio represents the number of molecule of interest (in a specific list) divided by the total number of molecules in the pathway. Ref: IPA 7.0; Ingenuity Systems, Mountain View, CA, web site: http://www.ingenuity.com/products/pathways_analysis.html.

Fig.3. Gene tree clustering analysis of 370 β-arrestin2-regulated sequences in response to i.PTH

This analysis grades the gene expression profiles according to their resemblance, with short branches indicating more similar profiles and long branches less similar profiles. The colorscale is according to the scaled signal of Affymetrix microarray experiment (mean genes expression value equal 1.0 for each GeneChip, in yellow). Higher than mean gene expression tends to the red color, whereas lower than mean gene expression tends to the blue color. This gene tree cluster was generated with the GeneSpring GX 7.3 software (Silicon Genetics, Agilent Technologies AG, Basel).

Fig.4. Gene Ontology (GO) categories of 370 β-arrestin2-regulated sequences in response to i.PTH

(A) GO categories by Molecular functions (ref. GO:3674). (B) GO categories by Cellular processes (ref. GO:9987). Numbers in parenthesis represent the number of sequences found in the respective sub-categories. These pie charts were generated with the GeneSpring GX 7.3 software (Silicon Genetics, Agilent Technologies AG, Basel).

Fig.5. Principal networks of β-arrestin2-regulated genes in response to i.PTH

(A) Network based on the 38 genes up- or down-regulated by i.PTH in presence/absence of β-arrestin2, centered on p38 MAPK and NFκB genes. (B) Network based on the 6 genes regulated by i.PTH in absence βarrestin2, centered on TGFB1. Closed symbols represent differentially regulated genes identified in the microarray analysis (A: Arrb2, Cebpd, Fgf7, Mid2, Omd, Pla2g7, Relb, Sdc4, Steap4, Ttc3 and Unc93b1, B: Rnf11, Serpinb1, Sdc4, Sox4 and Vasn). For gene name description see Table 2. Boxes contain the legends for relationship between genes and network shapes. Ref: IPA 7.0; Ingenuity Systems, Mountain View, CA, web site: http://www.ingenuity.com/products/pathways_analysis.html.