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.
"AMP-activated protein kinase AMPK ↓ MC3T3-E1 cell line and primary mouse calvarial osteoblasts  BMP-2-inducible kinase BIK ↓ MC3T3-E1 cell line  Calmodulin-dependent kinase II CaMKII ↑ C2C12 cell line  Casein kinase 2 CK2 ↓ C2C12 cell lines  Proto-oncogene tyrosine–protein kinase Src c-Src ↓ IL-6 TG mouse and primary mouse calvarial osteoblasts  Extracellular signal-regulated kinase 1/2 ERK1/2 ↑ ERK1/2 KO mouse & C3H10T1/2 cell line    Focal adhesion kinase FAK ↑ hBMSC   Glycogen synthase kinase 3 beta GSK3B ↓ Primary mouse bone marrow cells, C2C12, and MC3T3 cell lines  Jun N-terminal kinase JNK ↑ MC3T3-E1 cell line   Jun N-terminal kinase JNK ↓ MC3T3-E1 cell line & primary mouse BMSC   LIM domain kinase 2 LIMK2 ↑ Primary calvarial osteoblasts  Mixed-lineage kinase 3 MLK3 ↑ MLK3 KO mouse  Nemo-like kinase NLK ↓ Primary calvarial osteoblasts and ST2 cell line   p38 mitogen-activated protein kinase P38 MAPK ↑ P38β KO mouse, primary mouse calvarial osteoblasts, C2C12 cell line [126,154–156] Protein kinase A PRKA ↑ Human BMSC  Protein kinase A PRKA ↓ Mouse calvarial osteoblasts, mouse BMSC, rat BMSCs, and MC3T3-E1 cell line  Protein kinase C PRKC ↓ C2C12 and MC3T3-E1 cell lines   Protein kinase A PRKG1 ↓ Human BMSCs Unpublished data Protein tyrosine kinase 7 PTK7 ↓ Human BMSCs Unpublished data Proline-rich tyrosine kinase 2 Pyk2 ↓ Pyk2 KO mouse and human BMSC  Rho-associated, coiled-coil containing protein kinase ROCK ↑ Human BMSC, and MC3T3-E1, and C3H10T1/2 cell line [131–134,162] TGF-β-activated kinase 1 TAK1 ↑ TAK1 KO mouse  Future perspectives Dlk1/Pref-1, Lrp5/Wnt signaling and intracellular kinases are among the current themes in hBMSC and osteoblast biology. They are also examples of the power of combining molecular biology with genetic studies in vivo to unravel novel mechanisms controlling BMSC differentiation into osteoblastic cells and represent approaches to identify druggable targets for in vivo enhancing bone formation. "
[Show abstract][Hide abstract] ABSTRACT: Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSCs differentiation into osteoblastic cells is a strategy for identifying druggable targets for enhancing bone formation. This review will discuss the functions and the molecular mechanisms of action on osteoblast differentiation and bone formation; of a number of recently identified regulatory molecules: the non-canonical Notch signaling molecule Delta-like 1/preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases.
Bone 08/2014; 70. DOI:10.1016/j.bone.2014.07.028 · 3.97 Impact Factor
"Furthermore, SETDB1 (SET domain bifurcated 1), a histone methyltransferase, is phosphorylated by NLK, upon Wnt5a stimulation. Phosphorylation of SETDB1 leads to disruption of the PPAR-gamma function through methylation, a mechanism shown to be vital for lineage decision of mesenchymal stem cells , , . "
[Show abstract][Hide abstract] ABSTRACT: Nemo-like kinase (NLK), a proline-directed serine/threonine kinase regulated by phosphorylation, can be localized in the cytosol or in the nucleus. Whether the localization of NLK can affect cell survival or cell apoptosis is yet to be disclosed. In the present study we found that NLK was mainly localized in the nuclei of breast cancer cells, in contrast to a cytosolic localization in non-cancerous breast epithelial cells. The nuclear localization of NLK was mediated through direct interaction with Heat shock protein 27 (HSP27) which further protected cancer cells from apoptosis. The present study provides evidence of a novel mechanism by which HSP27 recognizes NLK in the breast cancer cells and prevents NLK-mediated cell apoptosis.
PLoS ONE 05/2014; 9(5):e96506. DOI:10.1371/journal.pone.0096506 · 3.23 Impact Factor
[Show abstract][Hide abstract] 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.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.