Gs/Gi Regulation of Bone Cell Differentiation: Review and Insights from Engineered Receptors.

G-protein coupled receptors (GPCRs) and their ligands are critical for normal osteoblast formation and function. GPCRs mediate a wide variety of biological processes and are activated by multiple types of extracellular signals, ranging from photons to small molecules to peptides. GPCRs signal through a select number of canonical pathways: the Gs and Gi pathways increase or decrease intracellular cAMP levels, respectively, by acting on adenylate cyclase, while the Gq pathway increases intracellular calcium by activating phospholipase C. In addition, non-canonical GPCR pathways such as ß-arrestin activation are important for osteoblast function. Since many cells express multiple GPCRs, and each individual GPCR may activate multiple signaling pathways, the resulting combinatorial signal provides a mechanism for regulating complex biological processes and effector functions. However, the wide variety of GPCRs, the possibility of multiple receptors acting with signaling redundancy, and the possibility of an individual GPCR activating multiple signaling pathways, also pose challenges for elucidating the role of a particular GPCR. Here, we briefly review the roles of Gs and Gi GPCR signaling in osteoblast function. We describe the successful application of a strategy for directly manipulating the Gs and Gi pathways using engineered receptors. These powerful tools will allow further elucidation of the roles of GPCR signaling in specific lineages of osteoblastic cells, as well as in non-osteoblast cells, all of which remain critical areas of active research.

Mineral composition is altered by osteoblast expression of an engineered G(s)-coupled receptor.

Activation of the G(s) G protein-coupled receptor Rs1 in osteoblasts increases bone mineral density by 5- to 15-fold in mice and recapitulates histologic aspects of fibrous dysplasia of the bone. However, the effects of constitutive G(s) signaling on bone tissue quality are not known. The goal of this study was to determine bone tissue quality in mice resulting from osteoblast-specific constitutive G(s) activation, by the complementary techniques of FTIR spectroscopy and synchrotron radiation micro-computed tomography (SRµCT). Col1(2.3)-tTA/TetO-Rs1 double transgenic (DT) mice, which showed osteoblast-specific constitutive G(s) signaling activity by the Rs1 receptor, were created. Femora and calvariae of DT and wild-type (WT) mice (6 and 15 weeks old) were analyzed by FTIR spectroscopy. WT and DT femora (3 and 9 weeks old) were imaged by SRµCT. Mineral-to-matrix ratio was 25% lower (P = 0.010), carbonate-to-phosphate ratio was 20% higher (P = 0.025), crystallinity was 4% lower (P = 0.004), and cross-link ratio was 11% lower (P = 0.025) in 6-week DT bone. Differences persisted in 15-week animals. Quantitative SRµCT analysis revealed substantial differences in mean values and heterogeneity of tissue mineral density (TMD). TMD values were 1,156 ± 100 and 711 ± 251 mg/cm(3) (mean ± SD) in WT and DT femoral diaphyses, respectively, at 3 weeks. Similar differences were found in 9-week animals. These results demonstrate that continuous G(s) activation in murine osteoblasts leads to deposition of immature bone tissue with reduced mineralization. Our findings suggest that bone tissue quality may be an important contributor to increased fracture risk in fibrous dysplasia patients.

Characterization of growth-differentiation factor 15, a transforming growth factor beta superfamily member induced following liver injury.

We have identified a new murine transforming growth factor beta superfamily member, growth-differentiation factor 15 (Gdf15), that is expressed at highest levels in adult liver. As determined by Northern analysis, the expression of Gdf15 in liver was rapidly and dramatically up-regulated following various surgical and chemical treatments that cause acute liver injury and regeneration. In situ hybridization analysis revealed distinct patterns of Gdf15 mRNA localization that appeared to reflect the known patterns of hepatocyte injury in each experimental treatment. In addition, treatment of two hepatocyte-like cell lines with either carbon tetrachloride or heat shock induced Gdf15 mRNA expression, indicating that direct cellular injury can induce Gdf15 expression in the absence of other cell types, such as inflammatory cells. In order to investigate the potential functions of Gdf15, we created Gdf15 null mice by gene targeting. Homozygous null mice were viable and fertile. Despite the dramatic regulation of Gdf15 expression observed in the partial-hepatectomy and carbon tetrachloride injury models, we found no differences in the injury responses between homozygous null mutants and wild-type mice. Our findings suggest either that Gdf15 does not have a regulatory role in liver injury and regeneration or that Gdf15 function within the liver is redundant with that of other signaling molecules.

Structural and functional conservation of the Drosophila doublesex splicing enhancer repeat elements.

We have compared the RNA sequences and secondary structures of the Drosophila melanogaster and Drosophila virilis doublesex (dsx) splicing enhancers. The sequences of the two splicing enhancers are highly divergent except for the presence of nearly identical 13-nt repeat elements (six in D. melanogaster and four in D. virilis) and a stretch of nucleotides at the 5' and 3' ends of the enhancers. In vitro RNA structure probing of the two enhancers revealed that the 13-nt repeats are predominantly single-stranded. Thus, both the primary sequences and single-stranded nature of the repeats are conserved between the two species. The significance of the primary sequence conservation was demonstrated by showing that the two enhancers are functionally interchangeable in Tra-/Tra2-dependent in vitro splicing. In addition, inhibition of splicing enhancer activity by antisense oligonucleotides complementary to the repeats demonstrated the importance of the conserved single-stranded structure of the repeats. In vitro binding studies revealed that Tra2 interacts with each of the D. melanogaster repeat elements, except for repeat 2, with affinities that are indistinguishable, whereas Tra binds nonspecifically to the enhancer. Taken together, these observations indicate that the organization of sequences within the dsx splicing enhancers of D. melanogaster and D. virilis results in a structure in which each of the repeat elements is single-stranded and therefore accessible for specific recognition by the RNA-binding domain of Tra2.

Cytokine-responsive gene-2/IFN-inducible protein-10 expression in multiple models of liver and bile duct injury suggests a role in tissue regeneration.

IFN-inducible protein-10 (IP-10/CXCL10) is a CXC chemokine that targets both T cells and NK cells. Elevation of IP-10 expression has been demonstrated in a number of human diseases, including chronic cirrhosis and biliary atresia. Cytokine-responsive gene-2 (Crg-2), the murine ortholog of IP-10, was induced following CCl(4) treatment of the hepatocyte-like cell line AML-12. Crg-2 expression was noted in vivo in multiple models of hepatic and bile duct injury, including bile duct ligation and CCl(4), D-galactosamine, and methylene dianiline toxic liver injuries. Induction of Crg-2 was also examined following two-thirds hepatectomy, a model that minimally injures the remaining liver, but that requires a large hepatic regenerative response. Crg-2 was induced in a biphasic fashion after two-thirds hepatectomy, preceding each known peak of hepatocyte DNA synthesis. Induction of Crg-2 was also observed in the kidney, gut, thymus, and spleen within 1 h of two-thirds hepatectomy. Characteristic of an immediate early gene, pretreatment of mice with the protein synthesis inhibitor cycloheximide before either two-thirds hepatectomy or CCl(4) injection led to Crg-2 superinduction. rIP-10 was demonstrated to have hepatocyte growth factor-inducing activity in vitro, but alone had no direct mitogenic effect on hepatocytes. Our data demonstrate that induction of Crg-2 occurs in several distinct models of liver injury and regeneration, and suggest a role for CRG-2/IP-10 in these processes.