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.

Keywords: JAK2; Maxillary bone disease; Maxillary development; Non-canonical JAGGED1 signaling; Osteoblast commitment.

Figure 1:

JAGGED1 induces the NOTCH pathway, by inducing expression of Notch1 receptor in (A), and expression of classic NOTCH targets (B) Hes1 and (C) Hey1. The expression of all genes induced by JAG1 was observed to be inhibited by a canonical NOTCH pathway inhibitor, DAPT (red bar, red patterned bars). Dynabeads bound recombinant JAG1-Fc fragment (yellow) or BMP2 (green, positive control) were used. Growth media, osteogenic media, dynabeads alone and dynabeads bound Fc-fragment were used as controls (black bars). (n=3) (Similar symbols = no difference, different symbols = significant difference) (S.D p<0.05)

Figure 2:

JAGGED1 induces osteoblastogenesis, as demonstrated by measuring the (A) alkaline phosphatase activity of O9-1 cells when treated with dynabeads bound recombinant JAG1-Fc fragment (yellow) or BMP2 (green, positive control). Growth media, osteogenic media, dynabeads alone and dynabeads bound Fc-fragment were used as controls (black bars). The canonical NOTCH pathway inhibitor DAPT (red bar, red patterned bars) did not inhibit JAG1-induced gene expression of (B) early osteoblast marker, Runx2, (C) late osteoblast marker, Ocn. (n=3) (Similar symbols = no difference, different symbols = significant difference) (S.D p<0.05)

Figure 3:. JAGGED1 induces a non-canonical NOTCH pathway.

(A) Lysates obtained from O9-1 cells treated with dynabeads alone (entire blots in Supplemental Figure S4A) or bound to Fc-fragment, BMP2 and etoposide, both known inducers of JAK2 phosphorylation (data not shown) and dynabeads bound recombinant JAG1-Fc fragment, were probed for phosphorylated JAK2 (entire blots in Supplemental Figure S4B). (B) O9-1 cells were treated with a NOTCH canonical pathway inhibitor, DAPT, prior to adding treatments. Lysates were then probed for JAK2 phosphorylation.

Figure 4:

JAGGED1 induces the NOTCH canonical pathway, demonstrated by treatment of O9-1 cells with an inhibitor of JAK2 phosphorylation, WP1066 (brown bar) prior to treatments with dynabeads bound recombinant JAG1-Fc fragment (yellow-brown patterned bar) or BMP2 (green-brown patterned bar) Notch1 receptor expression in (A) was significantly decreased, although expression of classic NOTCH targets (B) Hes1 and (C) Hey1 were uninhibited in the presence of WP1066. Growth media, osteogenic media, dynabeads alone and dynabeads bound Fc-fragment were used as controls (black bars). (n=3) (Similar symbols = no difference, different symbols = significant difference) (S.D p<0.05)

Figure 5:

JAGGED1 induces osteoblastogenesis via NOTCH non canonical signal through phosphorylated JAK2, as demonstrated by measuring the gene expression of (A) early osteoblast marker, Runx2, (B) late osteoblast marker, Ocn, (C) alkaline phosphatase (ALP) activity of O9-1 cells treated with an inhibitor of JAK2 phosphorylation prior to treatment with dynabeads bound recombinant JAG1-Fc fragment (yellow-brown patterned bar) or BMP2 (green-brown patterned bar). Runx2 and Ocn were significantly decreased in JAG1 + WP1066-treated cells. ALP activity was significantly decreased in JAG1 + DAPT-treated (yellow-red patterned bar) as well as JAG1 + WP1066-treated O9-1 cells but it was synergistically decreased in O9-1 cells treated with JAG1 along with both DAPT and WP1066 (yellow-purple patterned bar). Growth media, osteogenic media, dynabeads alone and dynabeads bound Fc-fragment were used as controls (black bars). (n=3) (Similar symbols = no difference, different symbols = significant difference) (S.D p<0.05).

Figure 6:. Illustration of mechanism of JAGGED1-induced osteoblast commitment of neural crest cells.

JAG1 induces osteoblast formation not only by activating the canonical NOTCH pathway but also by activating a non-canonical NOTCH signal involving the phosphorylation of JAK2. Future directions include understanding (A) the role of the origin of the neural crest cells during intramembranous ossification, (B) action of JAG1 through other NOTCH receptors (–4), (C) upstream and (D) downstream targets of JAK2 as well as whether there can be (E) crosstalk between the canonical and non-canonical pathways during JAGGED1 induced osteoblast formation.