Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation

Abstract

Wnt signaling regulates a variety of developmental processes in animals. Although the beta-catenin-dependent (canonical) pathway is known to control cell fate, a similar role for noncanonical Wnt signaling has not been established in mammals. Moreover, the intracellular cascades for noncanonical Wnt signaling remain to be elucidated. Here, we delineate a pathway in which Wnt3a signals through the Galpha(q/11) subunits of G proteins to activate phosphatidylinositol signaling and PKCdelta in the murine ST2 cells. Galpha(q/11)-PKCdelta signaling is required for Wnt3a-induced osteoblastogenesis in these cells, and PKCdelta homozygous mutant mice exhibit a deficit in embryonic bone formation. Furthermore, Wnt7b, expressed by osteogenic cells in vivo, induces osteoblast differentiation in vitro via the PKCdelta-mediated pathway; ablation of Wnt7b in skeletal progenitors results in less bone in the mouse embryo. Together, these results reveal a Wnt-dependent osteogenic mechanism, and they provide a potential target pathway for designing therapeutics to promote bone formation.

Figure 1. Wnt3a induces osteoblast differentiation and MARCKS phosphorylation via PKCδ in ST2 cells

(A) AP detection by substrate staining after incubation for 48 hrs in conditioned medium. (B) Detection of bone nodules by alizarin red after incubation for 14 days in mineralization medium. (C) Real-time PCR assays of mRNA expressed as folds over GAPDH. (D) AP expression in cells cultured for 48 hrs in serum-free medium with recombinant Wnt3a. (E) Upregulation of MARCKS (arrows) by Wnt3a. (F) Western analyses of total MARCKS in whole cell lysates from cells incubated in Wnt3a (W) or L medium. (G) Western analyses of phospho-MARCKS in cytosolic fractions of cells. (H) Western analyses of phospho-MARCKS in cytosolic fractions of cells incubated in serum-free medium. Wnt3a used at 50 ng/ml, and rottlerin at 5 μM. Bar graphs: n=3. Western signals normalized to α-tubulin.

Figure 2. PKCδ is required for Wnt3a-induced osteoblastogenesis but not for canonical Wnt signaling in ST2 cells

(A) Effects of PKC inhibitors on Wnt3a-induced AP expression in cells incubated for 48 hrs in conditioned medium. (B) Wnt3a-induced AP expression, and inhibition by rottlerin in primary E13.5 limb primordial cells after 96 hrs of incubation. 1: L medium; 2: Wnt3a medium; 3: Wnt3a medium plus 5 μM rottlerin. (C) Effect of Ro-31-8220 on Wnt3a-induced expression of Lef1-luciferase reporter. (D) Western analyses of β-catenin in cytosolic fractions of cells following incubation in Wnt3a (W) or L medium, with or without 5 μM rottlerin (Rott) or Ro-31-8220 (Ro). When inhibitors were used, cells were pretreated with inhibitor for 1 hr in normal growth medium. Bar graphs: n=3. β-catenin level normalized to α-tubulin.

Figure 3. PKCδ activation via G_q signaling promotes Wnt3a-induced osteoblastogenesis and requires Dvl in ST2 cells

(A–D) AP expression in cells incubated for 48 hrs in conditioned medium. In A and C, cells transfected with siRNA were incubated in normal growth medium for 96 hrs before cultured in conditioned medium. In B, cells were first infected with retrovirus before incubated in conditioned medium. n=3. (E–F) Western analyses for PKCδ (E) and Gα_q/Gα₁₁ (F) in whole cell lysates of cells at 96 hrs after siRNA transfection, with GAPDH siRNA as control. (G–L) Detection of bone nodules by alizarin red. In G–J, cells were infected with retrovirus before incubated in Wnt3a-mineralization medium. U73122 used at 5 μM. (M–N) Western analyses for β-catenin and phospho-MARCKS in cytosolic fractions. Cells prestarved for 24 hrs were cultured in serum-free medium for 1 hr with or without recombinant Wnt3a. In N, cells were first infected with viruses, and then starved for 24 hrs before Wnt3a stimulation. Western signals normalized to α-tubulin.

Figure 4. PKCδ activation by Wnt3a correlates with Dvl-2 translocation to the plasma membrane and is insensitive to Dkk1 in ST2 cells

(A–H) Immunostaining of PKCδ and Dvl-2 in cells with (A–D) or without (E–H) Wnt3a stimulation. In D and H, cells were infected with a retrovirus co-expressing Dkk1 and nuclear GFP (Dkk1-GFP). (I–J) Western analyses in cytosolic fractions. Cells were prestarved in serum-free medium for 24 hrs before stimulated with recombinant Wnt3a for indicated times (I) or for 1 hr (J). In J, cells were first infected with retroviruses expressing either GFP or Dkk1 before starvation. Signal levels normalized to α-tubulin. (K) Effect of Dkk1 on Lef1-luciferase activation by Wnt3a.

Figure 5. Removal of PKCδ results in a deficit in embryonic bone formation

(A–E) Whole-mount skeletal staining of wild type and PKCδ^−/− littermates at E15.5. Bone stained red; cartilage stained blue. Vertical lines in E demarcate ends of bone collar; horizontal red line denote deficit in mutant. R: ribs; Mx: maxilla; Mb: mandible. (F) von Kossa staining on longitudinal sections of E14.5 humerus and E15.5 radius in wild type versus PKCδ^−/− littermates. Vertical lines demarcate ends of bone collar; horizontal red line denote deficit in mutant. Double-headed arrows indicate lengths of bone collar (x) and total radius (y). (G) Relative bone length (x/y) in radius of E15.5 wild type versus PKCδ^−/− embryos. n=4, p<0.001. (H) In situ hybridization for chondrocyte markers on longitudinal sections of humerus in wild type versus PKCδ^−/− E14.5 littermates. Vertical lines denote ends of Colα1(X)-expressing domain in wild type embryo. Double-headed arrows indicate wild-type distance between two major PTHrP-R and Ihh expression domains. Asterisk: PTHrP-R signal in skin; arrow: MMP13 signal in osteoblast-lineage cell. (I) In situ hybridization for osteoblast markers on longitudinal sections of tibia in wild type versus PKCδ^−/− littermates at E15.5. Adjacent sections used for each genotype. Purple vertical lines: leading edge of Colα1(X)-expressing domain; orange vertical lines: leading edge of Osx or Bsp in wild type embryo; green vertical lines: leading edge of Osx or Bsp in PKCδ^−/− embryo; red horizontal lines: deficit in PKCδ^−/− embryo. Arrows: signal in perichondrium. (J) Western analyses of cytosolic fractions of limb primordial cells from E14.5 wild type versus PKC ^−/− littermates. (K–N) Detection of bone (K–L) and cartilage (M–N) nodules in primary cultures of limb primordial cells from E13.5 wild type (K and M) versus PKCδ^−/− (L and N) embryos. Bone nodules stained dark red; cartilage nodules blue. Relative nodule numbers between normal and mutant genotypes were indicated. (O) AP expression by wild type versus PKCδ^−/− BMSC at 72 hrs after confluence.

Figure 6. Wnt7b induces osteoblastogenesis and PKCδ activity but does not activate canonical Wnt signaling

(A) Detection of bone nodules by alizarin red in ST2 and C3H10T1/2 cells expressing GFP or Wnt7b. (B–C) Lef1-luciferase activation by Wnt3a, Wnt7b or daβcat. In B, Wnt7b was virally expressed whereas Wnt3a was present in conditioned medium. In C, cells were co-transfected with the reporter and a daβcat- or Wnt7b- expressing plasmid, or the empty expression vector pCIG. n=3. (D–E) Western analyses in cytosolic fractions of ST2 (D) or C3H10T1/2 (E) cells virally expressing GFP or Wnt7b, after incubation in serum-free medium for 24 hrs. Western signals normalized to GAPDH. (F–H) AP expression in C3H10T1/2 (F and G) or primary cultures of E13.5 limb primordial cells (H). Cells either transiently transfected (F) or virally infected (G–H) were cultured in normal growth medium for 48 hrs (F–G) or 72 hrs (H). n=3.

Figure 7. Removal of Wnt7b in skeletal cells results in defects in bone formation

(A) Whole-mount skeletal staining of wild type (WT) or Wnt7b mutant (MT1 and MT2) littermates at E15.5. Note smaller size of MT2. Representative bones shown at a higher magnification below corresponding whole skeleton. R: ribs; Mx: Maxilla; Mb: mandible; s: scapula; h: humerus; c: clavicle. (B) von Kossa staining on longitudinal sections of humerus in wild type (WT) versus Wnt7b mutant (MT) E15.5 littermates. Vertical lines demarcate length of bone collars; horizontal line denotes deficit in mutant embryo. (C) Relative bone length (bone collar over total length) in humerus of wild type versus Wnt7b mutant E14.5 littermates. n=5; p<0.001. (D) Skulls of wild type (WT) or Wnt7b mutant (MT) littermates at E18.5. Boxed regions shown at a higher magnification below. White contour demarcates sutures; arrow denotes a nearly fused suture. (E) Western analyses of cytosolic fractions of limb primordial cells from wild type versus Wnt7b mutant E14.5 littermates. (F) In situ hybridization for chondrocyte markers on longitudinal sections of humerus from wild type (WT) versus Wnt7b mutant (MT) embryos. Vertical lines denote ends of Colα1(X)-expressing domain in wild type embryo. Double-headed arrows indicate wild-type distance between the two major PTHrP-R, Ihh or Colα1(X) expression domains. Asterisk: PTHrP-R signal in skin. (G) In situ hybridization for osteoblast markers on longitudinal sections of humerus in wild type versus Wnt7b mutant (MT) E15.5 littermates. Adjacent sections were used for each genotype. Purple vertical lines: leading edge of Colα1(X)-expressing domain in each genotype; orange vertical lines: leading edge of Osx, PTHrP-R or Bsp in wild type; green vertical lines: leading edge of Osx, PTHrP-R or Bsp in MT embryo; red horizontal lines: deficit in MT embryo. Arrows: signal in perichondrium; asterisks: signal in chondrocytes. (H) Detection of bone nodules in primary cultures of calvarial cells or bone marrow stromal cells (BMSC) from wild type (WT) or Wnt7b mutant (MT) littermates. (I) In situ hybridization for Dkk1 and Tcf1 on longitudinal sections of humerus at E15.5. Asterisks: signal in chondrocytes; arrows: signal in perichondrium; PH: prehypertrophic chondrocytes. (J) Wnt7b signals through a Gα_q/11→PLC→PKCδpathway to stimulate progression from Runx2- to Osx-expressing cells during osteoblastogenesis.