Activation of the ERK pathway in osteoblastic cells, role of gremlin and BMP-2

Gremlin is a glycoprotein that binds and antagonizes the actions of bone morphogenetic proteins (BMPs) -2, -4, and -7. Gremlin appears to activate the extracellular regulated kinase (ERK) pathway in endothelial and tumor cells, and as a consequence to have direct cellular effects. To determine whether gremlin has direct effects in osteoblasts, independent of its BMP binding activity, we examined its effects in ST-2 murine stromal cell lines and in primary cultures of murine calvarial osteoblasts. Gremlin did not activate Signaling mothers against decapentaplegic (Smad), and suppressed the BMP-2 induced Smad 1/5/8 phosphorylation and the transactivation of the BMP/Smad reporter construct 12xSBE-Oc-pGL3, confirming its BMPs antagonizing activity. Neither gremlin nor BMP-2 induced ERK 1/2 activation in ST-2 cells or calvarial osteoblasts. Moreover, slight changes in culture conditions induced the phosphorylation of ERK independent from BMP or gremlin exposure. In conclusion, gremlin inhibits BMP-2 signaling and activity, and does not have independent actions on ERK signaling in osteoblasts. Consequently, gremlin activity in osteoblasts can be attributed only to its BMP antagonizing effects.     

Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3'-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3'-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3'-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation.     

A 32-kilodalton protein binds to AU-rich domains in the 3'' untranslated regions of rapidly degraded mRNAs.

An AU-rich sequence present within the 3' untranslated region has been shown to mark some short-lived mRNAs for rapid degradation. We demonstrate by label transfer and gel shift experiments that a 32-kDa polypeptide, present in nuclear extracts, specifically interacts with the AU-rich domains present within the 3' untranslated region of human granulocyte-macrophage colony-stimulating factor, c-fos, and c-myc mRNAs and a similar domain downstream of the poly(A) addition site of the adenovirus IVa2 mRNA. Competition experiments and partial protease analysis indicated that the same polypeptide interacts with all four RNAs. A single AUUUA sequence in a U-rich context was sufficient to signal binding of the 32-kDa polypeptide. Insertion of three copies of this minimal recognition site led to markedly reduced accumulation of beta-globin RNA, while the same insert carrying a series of U-to-G changes had little effect on RNA levels. Steady-state levels of beta-globin-specific nuclear RNA, including incompletely processed RNA, and cytoplasmic mRNA were reduced. Cytoplasmic mRNA containing the AU-rich recognition sites for the 32-kDa polypeptide exhibited a half-life shorter than that of mRNA with a mutated insert. We suggest that binding of the 32-kDa polypeptide may be involved in the regulation of mRNA half-life.     

Tumor necrosis factor alpha-induced glucose transporter (GLUT-1) mRNA stabilization in 3T3-L1 preadipocytes. Regulation by the adenosine-uridine binding factor.

Tumor necrosis factor alpha (TNF alpha), 12-O-tetradecanoylphorbol-13-acetate and cAMP stimulate hexose transport in quiescent 3T3-L1 preadipocytes by stabilizing the relatively labile mRNA coding for the basal glucose transporter, GLUT-1. The 3'-UTR of GLUT-1 mRNA contains a single copy of the destabilizing AUUUA motif in the context of an AU-rich region. The adenosine-uridine binding factor (AUBF) is a cytosolic protein which interacts with similar AU-rich regions in a variety of labile cytokine and oncogene mRNAs. Here, we demonstrate that AUBF complexes in vitro with GLUT-1 mRNA through the AU-rich portion of the 3'-UTR. AUBF activity is very low in quiescent preadipocytes, but can be up-regulated by agonists such as TPA, TNF alpha, cAMP, and okadaic acid, all of which stabilize GLUT-1 mRNA. The time courses of TNF alpha- and TPA-mediated AUBF up-regulation and GLUT-1 mRNA stabilization are coincident, suggesting a cause and effect relationship.     

Conditional deletion of gremlin causes a transient increase in bone formation and bone mass

Gremlin is a glycoprotein that binds bone morphogenetic proteins (BMPs) 2, 4, and 7, antagonizing their actions. Gremlin opposes BMP effects on osteoblastic differentiation and function in vitro and in vivo, and its overexpression causes osteopenia. To define the function of gremlin in the skeleton, we generated gremlin 1 (grem1) conditional null mice by mating mice where grem1 was flanked by lox(P) sequences with mice expressing the Cre recombinase under the control of the osteocalcin promoter. grem1 null male mice displayed increased trabecular bone volume due to enhanced osteoblastic activity, because mineral apposition and bone formation rates were increased. Osteoblast number and bone resorption were not altered. Marrow stromal cells from grem1 conditional null mice expressed higher levels of alkaline phosphatase activity. Gremlin down-regulation by RNA interference in ST-2 stromal and MC3T3 osteoblastic cells increased the BMP-2 stimulatory effect on alkaline phosphatase activity, on Smad 1/5/8 phosphorylation, and on the transactivation of the BMP/Smad reporter construct 12xSBE-Oc-pGL3. Gremlin down-regulation also enhanced osteocalcin and Runx-2 expression, Wnt 3a signaling, and activity in ST-2 cells. In conclusion, deletion of grem1 in the bone microenvironment results in sensitization of BMP signaling and activity and enhanced bone formation in vivo.     

The AU-rich 3' untranslated region of human MDR1 mRNA is an inefficient mRNA destabilizer.

The human multidrug resistance gene MDR1 encodes a membrane-bound protein, referred to as P-glycoprotein, that acts as a pump to extrude toxins from cells. The 3' untranslated region (3'UTR) of the human MDR1 mRNA is very AU-rich (70%) and contains AU-rich sequences similar to those shown to confer rapid decay on c-myc, c-fos, and lymphokine mRNAs. We tested the ability of the MDR1 3'UTR to act as an mRNA destabilizing element in the human hepatoma cell line HepG2. The MDR1 mRNA has an intermediate half-life of 8 h in HepG2 cells compared to a half-life of 30 min for c-myc mRNA. The MDR1 mRNA half-life was prolonged to >20 h upon treatment with the protein synthesis inhibitor cycloheximide. We constructed expression vectors containing the human beta-globin coding region with the 3'UTR from either MDR1 or c-myc. The c-myc 3'UTR increased the decay of the chimeric mRNA, but the MDR1 3'UTR had no effect. We tested the ability of MDR1 3'UTR sequences to compete for interaction with AU-binding proteins in cell extracts; MDR1 RNA probes had a fivefold lower affinity for AU-binding proteins that interact with the c-myc AU-rich 3'UTR. Overall, our data suggest that the MDR1 3'UTR does not behave as an active destabilizing element in HepG2 cells.