Stat3 loss in mesenchymal progenitors causes Job syndrome-like skeletal defects by reducing Wnt/β-catenin signaling

Abstract

Job syndrome is a rare genetic disorder caused by STAT3 mutations and primarily characterized by immune dysfunction along with comorbid skeleton developmental abnormalities including osteopenia, recurrent fracture of long bones, and scoliosis. So far, there is no definitive cure for the skeletal defects in Job syndrome, and treatments are limited to management of clinical symptoms only. Here, we have investigated the molecular mechanism whereby Stat3 regulates skeletal development and osteoblast differentiation. We showed that removing Stat3 function in the developing limb mesenchyme or osteoprogenitor cells in mice resulted in shortened and bow limbs with multiple fractures in long bones that resembled the skeleton symptoms in the Job Syndrome. However, Stat3 loss did not alter chondrocyte differentiation and hypertrophy in embryonic development, while osteoblast differentiation was severely reduced. Genome-wide transcriptome analyses as well as biochemical and histological studies showed that Stat3 loss resulted in down-regulation of Wnt/β-catenin signaling. Restoration of Wnt/β-catenin signaling by injecting BIO, a small molecule inhibitor of GSK3, or crossing with a Lrp5 gain of function (GOF) allele, rescued the bone reduction phenotypes due to Stat3 loss to a great extent. These studies uncover the essential functions of Stat3 in maintaining Wnt/β-catenin signaling in early mesenchymal or osteoprogenitor cells and provide evidence that bone defects in the Job Syndrome are likely caused by Wnt/β-catenin signaling reduction due to reduced STAT3 activities in bone development. Enhancing Wnt/β-catenin signaling could be a therapeutic approach to reduce bone symptoms of Job syndrome patients.

Keywords: Job syndrome; Sost; Stat3; Wnt/beta-catenin signaling; bone development.

Fig. 1.

Stat3 is required for bone development. (A) Gross appearance of 4-wk-old Stat3^C/C, Prrx1Cre: Stat3^C/+, and Prrx1Cre: Stat3^C/C (Stat3 knockout [KO]) mice (from Left to Right). (B and C) Quantification of body length (B) and weight (C) of 4-wk-old mice of the indicated genotypes. (D) Whole-mount Alizarin red and Alcian blue staining of Stat3 KO and littermate control mice at indicated ages. The forelimb (FL) and hindlimb (HL) were shown in the Lower. (Scale bars, 1 mm.) Bone fractures were indicated by red arrowheads. (E) Representative µCT images of FL of 5-wk-old littermate mice with the indicated genotypes. (Scale bars, 1 mm.) ***P < 0.001 and ****P < 0.0001 were considered significant; ns, not significant; the data are presented as mean ± SD.

Fig. 2.

Stat3 is required in limb mesenchymal cells for osteoblast differentiation and bone formation. (A–E) Representative images of Sp7 IF staining (A), Col1a1 in situ hybridization (B), Opn IF staining (C), von Kossa staining (D), and Mmp13 in situ hybridization (E) of the humerus sections from E16.5 littermate embryos or P0 littermate pups with indicated genotypes. The arrows indicate reduced osteoblast marker (protein/gene) expression or ossification. The arrowheads indicate bone fractures in the P0 mutant humerus. (Scale bars, 100 µm.) (F) Western blotting analyses of the humerus bone tissue lysates of the P0 pups with indicated genotypes. (F’) Quantification of the Western blotting results in (F). (G) Representative µCT images of femurs from 5-wk-old littermate mice with the indicated genotypes. (Scale bars, 1 mm.) (H) Quantification of humerus length (n = 3, mean ± SD). (I and J) Representative µCT images of the cortical (I) and trabecular (J) femur bones from 5-wk-old littermate mice. (Scale bars, 100 µm.) (K–M) Quantification of indicated parameters of µCT scanning. (N–P) Histomorphometric analysis of bone formation from 5-wk-old littermate mice of indicated genotypes. Representative images of double Calcein labeling in the distal femur heads (N), cortical bones (O), and trabecular bones (P) of the indicated genotypes. (Scale bars, 100 µm.) (Q) Quantification of indicated histomorphometric parameters of the distal femurs from 5-wk-old littermate mice of indicated genotypes. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered significant; ns, not significant. The data are shown as means ± SD.

Fig. 3.

Loss of Stat3 in osteoprogenitor cells phenocopied the Prrx1Cre; Stat3 mice. (A) Whole-mount Alizarin red and Alcian blue staining of the indicated littermate mouse embryos or pups. The forelimb (FL) and hindlimb (HL) were shown in the Lower. Bone fracture is shown by arrows. (Scale bars, 1 mm.) (B) Enhanced curvature of spine and multiple fractures (arrows) in the ribcage of P10 mutant mice. (C) Poorly developed and less-mineralized spine bones of P10 mutant mice. The T9 to T11 vertebras of the littermate animals with indicated genotypes are shown. The arrows indicate the defects in vertebras. (Scale bars, 1 mm.) (D and E) Representative in situ hybridization images of indicated gene expression or Sp7::GFP florescent images in the humerus sections of E16.5 (D) and E18.5 (E) littermate embryos with indicated genotypes. (Scale bars, 100 µm.)

Fig. 4.

Stat3 deletion leads to reduction in Wnt/β-catenin signaling activity in the developing long bones. (A) Schematics of the procedure for isolating E16.5 humerus bones for the RNA-seq analysis. (B) Heatmap analysis of differentially expressed genes between Control and Prrx1Cre; Stat3^c/c KO Samples. (C) GO enrichment analysis of the differentially expressed genes for biological processes revealed genes associated with skeleton development, osteoblast differentiation, and endochondral ossification (boxed in green). (D) GO enrichment analysis of down-regulated genes for signaling pathways revealed reduction in Wnt, TGF-β, Hedgehog, and Hippo signaling pathways (boxed in blue). (E) IF images of β-catenin expression in the humerus sections of littermate control and Stat3 KO P0 pups. (Scale bars, 100 µm.) (F) Western blotting analysis of β-catenin in the bone tissue lysates prepared from P0 pups with indicated genotypes. (G) Quantification of the Western blotting results in (F). (H) qRT-PCR analysis of Wnt/β-catenin signaling target genes in the humerus bone from the P0 littermate pups. *P < 0.05 and **P 0.005 were considered as significant; the data are presented as mean ± SD.

Fig. 5.

Skeleton defects in Prrx1Cre; Stat3^c/c mice were rescued by BIO treatment. (A) Whole-mount Alizarin red and Alcian blue staining of littermate mice of the indicated genotypes at P0, P5, and P14. The forelimb (FL) and hindlimb (HL) are shown in the Lower. (B–D) Representative µCT images of humerus (B) and cross-sections of the cortical (C) and trabecular (D) distal femur bones from 5-wk-old littermate mice of the indicated genotypes. (Scale bars, 1 mm in B and 100 µm in C and D.) (E) Quantification of humerus length (mean ± SD) and µCT parameters in femur bones from 5-wk-old littermate mice of the indicated genotypes. *P < 0.05, **P < 0.005, and ***P < 0.001. The data are shown as means ± SD. ns, not significant. (F) Representative images of fluorescent staining of Sp7, Opn, and Col1a1 in situ hybridization in the humerus sections of P0 littermate pups with indicated genotypes. (Scale bars, 100 µm.) Quantification was shown on the Right.

Fig. 6.

The Lrp5 high bone mass (HBM) allele (Lrp5^A/+) partially rescued the skeletal defects of the Prrx1Cre; Stat3^c/c mice. (A) Whole-mount Alizarin red and Alcian blue staining of littermate mice of the indicated genotypes at P0 and P5. The forelimb (FL) and hindlimb (HL) were shown in the Lower. (Scale bars, 100 µm.) (B) IF images of β-catenin and Sp7 expression in the humerus sections of P0 pups of the indicated genotypes. Higher-magnification images are shown on the Right side. (Scale bars, 100 µm.) Quantification was shown on the Right. *P < 0.05 and **P < 0.005. (C) Representative IF images of Mmp13 in humerus sections from the indicated P5 mice. DAPI stained the nucleus. n = 3 biological replicates. (Scale bar, 50 μm.) (D) von Kossa staining of the humerus sections of P0 pups of the indicated genotypes. Higher-magnification images are shown on the Right side. (Scale bars, 100 µm.)