Reciprocal Regulation of Notch and Nuclear Factor of Activated T-cells (NFAT) c1 Transactivation in Osteoblasts

Abstract

Notch proteins are single pass transmembrane receptors that play a critical role in cell fate decisions. Notch regulates several developmental processes, and contributes to the regulation of cell renewal in multiple organs and cell systems (1). Notch is involved in skeletal development and homeostasis, because it inhibits chondrocyte proliferation and differentiation, and suppresses osteoblast differentiation and osteoclastogenesis (2-6). There are four Notch receptors (Notch1 to 4) and five canonical ligands, which are Serrate/Jagged1 and 2, and Delta-Like 1, 3, and 4 (7). Notch-ligand interactions result in the proteolytic cleavage and release of the Notch intracellular domain (NICD) 2 (8). In the canonical Notch signaling pathway, NICD translocates to the nucleus and interacts with Epstein-Barr virus latency C promoter binding factor 1, suppressor of hairless and Lag-1 (CSL), which is bound to DNA and suppresses gene expression by recruiting transcriptional co-repressors. The binding of NICD to CSL induces the formation of a ternary complex with Mastermind-like proteins, which displaces the transcriptional co-repressors and recruits co-activators of transcription (9). These events induce the expression of Hairy Enhancer of Split (Hes) and Hairy/HES related with YRPF motif (Hey) transcription factors (10 -13).Nuclear factors of activated T-cells (NFAT) are five transcription factors (NFATc1 to c4 and NFAT5) involved in vertebrate development and in the growth and differentiation of multiple cell types (14 -17). In unstimulated cells, NFATc1 to c4 are highly phosphorylated and reside in the cytoplasm. Activation of the phosphatase calcineurin dephosphorylates specific serine residues in the SRR and SPXX repeat motifs of the regulatory domain of NFAT. This induces NFAT translocation to the nucleus, and activation of transcription of NFAT target genes (18). NFAT phosphorylation by protein kinases, such as glycogen synthase kinase 3␤ (GSK3␤), induces the nuclear export of NFAT preventing its transactivation (19 -21). Activity of GSK3␤ is suppressed by phosphorylation on serine 9, which is a target of protein kinases, such as cGMPdependent protein kinase II (cGKII), the product of the protein kinase cGMP-dependent type II (Prkg2) gene (22). cGKII activity is induced by increased cGMP levels and it is sustained by autophosphorylation on serine 126 (23).The fate of mesenchymal cells and their differentiation toward cells of the osteoblastic lineage is controlled by a network of extracellular and intracellular signals (24,25). We have reported that the overexpression of NICD in vitro inhibits osteoblast differentiation and that its overexpression in vivo causes osteopenia by reducing osteoblast number (5,26,27). Accordingly, the conditional deletion of Notch1 and Notch2 in the skeleton increases bone volume and induces the

Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption

“…5). In accordance with the histomorphometric findings revealing no changes in osteoblast number or function in 1-month-old Notch2 Q2319X mice, Notch2 Q2319X mutant cells expressed no significant changes in Bglap and Alpl mRNA levels (7,17). In accordance with the increase in bone resorption observed in Notch2 Q2319X mice, expression of Tnfsf11 was increased in osteoblast- (Fig.…”

“…This canonical signaling pathway leads to the transcription of Hairy Enhancer of Split (Hes)1, -5, and -7 and Hes related with YRPW motif (Hey)1, -2, and -L. Skeletal cells express Notch1, Notch2, and low levels of Notch3 transcripts (13)(14)(15). Activation of Notch in undifferentiated and differentiated osteoblasts inhibits cell differentiation and function and causes osteopenia (16,17). In contrast, activation of Notch1 in osteocytes causes a pronounced increase in bone mass due to a suppression of bone resorption (18).…”

Loss of Notch3 Signaling Enhances Osteogenesis of Mesenchymal Stem Cells from Mandibular Torus

“…Furthermore, recent studies have shown that Notch inhibited ATF4 transactivation and NFATc1 transcription, while NFATc1 reciprocally inhibited the transactivation of Notch target genes, suggesting that Notch and NFATc1 form a regulatory network to regulate the differentiation of osteoblasts (Zanotti et al 2011). Herein, we showed that T-MSCs constitutively express a higher level of ATF4 and NFATc1 than JB-MSCs, which is conversely correlated with the decreased expression of Notch3 in T-MSCs; knockdown of Notch3 expression led to increased expression of ATF4 and NFATc1 in JB-MSCs.…”

NFAT Signaling and Bone Homeostasis

“…Hey1 bond to the promoter of NFATc1 and suppressed its expression. Expression of NFATc1 was increased in the bone of RBPjk deleted mice [81]. These results indicate that Notch signaling suppresses NFATc1 in osteoblasts to prevent excessive bone formation.…”

“…Twist, as a negative regulator of osteoblast, binds to RUNX2 and inhibits its ability to activate target genes. Notch and CCL8 and many other proteins not described here also regulate osteoblasts [80][81][82].…”