Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation.
ABSTRACT Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.
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ABSTRACT: The bone morphogenetic protein/Signaling mothers against decapentaplegic (BMP/Smad) and the WNT signaling pathways regulate the commitment of mesenchymal cells to the osteoblastic lineage. Nemo-like kinase (Nlk) is an evolutionary conserved kinase that suppresses Smad transactivation and WNT canonical signaling. However, it is not clear whether these effects of Nlk have any consequence on the differentiation of mammalian cells. To study the function of Nlk during the commitment of ST-2 bone marrow stromal cells to the osteoblastic fate, Nlk was downregulated by RNA interference (RNAi), following transfection of a specific small interfering (si)RNA. Nlk downregulation increased alkaline phosphatase and osteocalcin expression and sensitized ST-2 cells to the effects of BMP2 and WNT3 on alkaline phosphatase mRNA expression and activity. Accordingly, Nlk downregulation enhanced the effect of BMP2 on the transactivation of the BMP/Smad reporter construct 12xSBE-Oc-pGL3, and on the levels of phosphorylated Smad1/5/8, whereas it did not affect the transactivation of the transforming growth factor-β/Smad reporter pSBE-Luc. Nlk downregulation sensitized ST-2 cells to the effects of WNT3 on the transactivation of the WNT/T-cell factor (Tcf) reporter construct 16xTCF-Luc, whereas it did not affect cytosolic β-catenin levels. To understand the function of Nlk in cells committed to the osteoblastic lineage, Nlk was suppressed by RNAi in primary calvarial osteoblasts. Downregulation of Nlk increased alkaline phosphatase and osteocalcin transcripts and sensitized osteoblasts to the effects of BMP2 on alkaline phosphatase activity and Smad1/5/8 transactivation and phosphorylation. In conclusion, Nlk suppresses osteoblastogenesis by opposing BMP/Smad and WNT canonical signaling.Journal of Cellular Biochemistry 09/2011; 113(2):449-56. · 3.06 Impact Factor
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ABSTRACT: Peroxisome-proliferator activator receptor γ (PPARγ) is a nuclear receptor of central importance in energy homeostasis and inflammation. Recent experimental pieces of evidence demonstrate that PPARγ is implicated in the oxidative stress response, an imbalance between antithetic prooxidation and antioxidation forces that may lead the cell to apoptotic or necrotic death. In this delicate and intricate game of equilibrium, PPARγ stands out as a central player devoted to the quenching and containment of the damage and to foster cell survival. However, PPARγ does not act alone: indeed the nuclear receptor is at the point of interconnection of various pathways, such as the nuclear factor erythroid 2-related factor 2 (NRF2), Wnt/β-catenin, and forkhead box proteins O (FOXO) pathways. Here we reviewed the role of PPARγ in response to oxidative stress and its interaction with other signaling pathways implicated in this process, an interaction that emerged as a potential new therapeutic target for several oxidative-related diseases.PPAR Research 01/2012; 2012:641087. · 2.69 Impact Factor
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ABSTRACT: Osteoporosis is a common polygenic disease and global healthcare priority but its genetic basis remains largely unknown. We report a high-throughput multi-parameter phenotype screen to identify functionally significant skeletal phenotypes in mice generated by the Wellcome Trust Sanger Institute Mouse Genetics Project and discover novel genes that may be involved in the pathogenesis of osteoporosis. The integrated use of primary phenotype data with quantitative x-ray microradiography, micro-computed tomography, statistical approaches and biomechanical testing in 100 unselected knockout mouse strains identified nine new genetic determinants of bone mass and strength. These nine new genes include five whose deletion results in low bone mass and four whose deletion results in high bone mass. None of the nine genes have been implicated previously in skeletal disorders and detailed analysis of the biomechanical consequences of their deletion revealed a novel functional classification of bone structure and strength. The organ-specific and disease-focused strategy described in this study can be applied to any biological system or tractable polygenic disease, thus providing a general basis to define gene function in a system-specific manner. Application of the approach to diseases affecting other physiological systems will help to realize the full potential of the International Mouse Phenotyping Consortium.PLoS Genetics 08/2012; 8(8):e1002858. · 8.52 Impact Factor