A non-canonical JAGGED1 signal to JAK2 mediates osteoblast commitment in cranial neural crest cells

Abstract

During craniofacial development, cranial neural crest (CNC) cells migrate into the developing face and form bone through intramembranous ossification. Loss of JAGGED1 (JAG1) signaling in the CNC cells is associated with maxillary hypoplasia or maxillary bone deficiency (MBD) in mice and recapitulates the MBD seen in humans with Alagille syndrome. JAGGED1, a membrane-bound NOTCH ligand, is required for normal craniofacial development, and Jagged1 mutations in humans are known to cause Alagille Syndrome, which is associated with cardiac, biliary, and bone phenotypes and these children experience increased bony fractures. Previously, we demonstrated deficient maxillary osteogenesis in Wnt1-cre;Jagged1 f/f (Jag1CKO) mice by conditional deletion of Jagged1 in maxillary CNC cells. In this study, we investigated the JAG1 signaling pathways in a CNC cell line. Treatment with JAG1 induced osteoblast differentiation and maturation markers, Runx2 and Ocn, respectively, Alkaline Phosphatase (ALP) production, as well as classic NOTCH1 targets, Hes1 and Hey1. While JAG1-induced Hes1 and Hey1 expression levels were predictably decreased after DAPT (NOTCH inhibitor) treatment, JAG1-induced Runx2 and Ocn levels were surprisingly constant in the presence of DAPT, indicating that JAG1 effects in the CNC cells are independent of the canonical NOTCH pathway. JAG1 treatment of CNC cells increased Janus Kinase 2 (JAK2) phosphorylation, which was refractory to DAPT treatment, highlighting the importance of the non-canonical NOTCH pathway during CNC cells osteoblast commitment. Pharmacologic inhibition of JAK2 phosphorylation, with and without DAPT treatment, upon JAG1 induction reduced ALP production and, Runx2 and Ocn gene expression.Collectively, these data suggest that JAK2 is an essential component downstream of a non-canonical JAG1-NOTCH1 pathway through which JAG1 stimulates expression of osteoblast-specific gene targets in CNC cells that contribute to osteoblast differentiation and bone mineralization.

JAGGED1 stimulates cranial neural crest cell osteoblast commitment pathways and bone regeneration independent of canonical NOTCH signaling

“…The copyright holder for this preprint this version posted June 24, 2020. ; https://doi.org/10.1101/2020.06.24.169755 doi: bioRxiv preprint We previously showed that JAG1 non-canonically signals through NOTCH via phosphorylation of JAK2 during CNC cell osteoblast commitment (8). In this manuscript, we demonstrated increased STAT5 phosphorylation, demonstrating that STAT5 (Fig.…”

“…Our prior research demonstrated that JAG1 signaling is required for CNC cell osteoblast commitment and that conditional ablation of Jag1 in the CNC cells of mice was associated with craniofacial bone loss, recapitulating the facial phenotype seen in Alagille Syndrome patients who have JAG1 mutations (4). JAG1 signaling occurs through cell-to-cell interactions between the JAG1 ligand and NOTCH receptors 1-4 (16)(17)(18) and we previously reported that JAG1-dependent CNC cell osteoblast commitment occurs primarily through non-canonical JAG1-JAK2 signaling (8).…”

“…Previously, our lab demonstrated that JAG1 can elicit a NOTCH non-canonical JAK2 signal which stimulates CNC cell osteoblast commitment (8), however the downstream mediators are unknown. To determine the non-canonical downstream mediators of JAG1-JAK2 signaling, CNC cells (n = 3) were treated in the presence and absence of the canonical NOTCH inhibitor DAPT (15 µM) with JAG1 (5.7 µM) and compared to CNC cells treated with BMP2 (100 nM) and Fc (5.7 µM) as positive and negative controls, respectively.…”

“…JAG1 is a membrane bound ligand of the NOTCH receptors 1-4 and signals via canonical (Hes1/Hey1) (4-7) and non-canonical (AKT, JAK2, MAPK and p38) intracellular pathways (8)(9)(10)(11)(12). JAG1 is required for normal craniofacial bone development in humans during which cranial neural crest (CNC) cells arise from the neural placode and migrate into the first brachial arch to form the calvarium and maxilla (4,13).…”

“…24.169755 doi: bioRxiv preprint NOTCH pathway. Based on our prior findings that JAG1 induces CNC cell differentiation into osteoblasts via a non-canonical NOTCH pathway mediated by phosphorylated JAK2 (8), we sought to determine the canonical vs non-canonical roles of JAG1-NOTCH signaling in CNC cells to identify potential novel therapeutic candidates for bone regeneration.…”