NO/cGMP signaling is important for bone remodeling in response to mechanical and hormonal stimuli, but the downstream mediator(s) regulating skeletal homeostasis are incompletely defined. We generated transgenic mice expressing a partly-activated, mutant cGMP-dependent protein kinase type 2 (PKG2R242Q) under control of the osteoblast-specific Col1a1 promoter to characterize the role of PKG2 in post-natal bone formation. Primary osteoblasts from these mice showed a two- to three-fold increase in basal and total PKG2 activity; they proliferated faster and were resistant to apoptosis compared to cells from WT mice. Male Col1a1-Prkg2R242Q transgenic mice had increased osteoblast numbers, bone formation rates and Wnt/β-catenin-related gene expression in bone and a higher trabecular bone mass compared to their WT littermates. Streptozotocin-induced type 1 diabetes suppressed bone formation and caused rapid bone loss in WT mice, but male transgenic mice were protected from these effects. Surprisingly, we found no significant difference in bone micro-architecture or Wnt/β-catenin-related gene expression between female WT and transgenic mice; female mice of both genotypes showed higher systemic and osteoblastic NO/cGMP generation compared to their male counterparts, and a higher level of endogenous PKG2 activity may be responsible for masking effects of the PKG2R242Q transgene in females. Our data support sexual dimorphism in Wnt/β-catenin signaling and PKG2 regulation of this crucial pathway in bone homeostasis. This work establishes PKG2 as a key regulator of osteoblast proliferation and post-natal bone formation.
Supplemental Figure 1: Activity of purified wild type and mutant PKG2 in vitro, presence of the prkg2RQ transgene in founder mice, and PKG2 protein level in POB membranes. (A,B) N-terminally Flag-tagged wild type and mutant PKG2 were affinity-purified from transfected 293T cells, and the proteins were analyzed by SDS-PAGE and Coomassie Blue staining (A). Kinase activity was measured with increasing concentrations of a synthetic peptide in the presence of 10 µM cGMP as described in Methods (B). (C) Presence of the prkg2RQ transgene was detected in tail DNA by PCR using primers F2/R2 shown in Fig. 1C; one male and two female founder mice with sex-matched non-transgenic litter mates are shown. The Fe-9.1 line was further characterized. (D) PKG2 protein was assessed by Western blotting of POB membrane fractions from two mice per genotype, with caveolin-1 serving as a loading control.
Supplemental Figure 2: Proliferation, ERK and Akt/GSK-3β signaling, and nuclear β-catenin in POBs isolated from 8 week-old female Col1a1-prkg2RQ transgenic and wild type mice. POBs were isolated from 8 week-old female transgenic mice and their wild type litter mates (n= 3 mice per genotype) and cultured as described for male POBs in Fig. 2. (A) Metabolically-active cells were quantified by measuring tetrazolium (MTS) reduction to formazan as described in Methods. (B) Serum-deprived POBs were treated with vehicle or 100 µM 8-CPT-cGMP (+ cGMP) for 10 min, and Western blots of cell extracts were analyzed for ERK1/2(pTyr204), Akt(pSer473), GSK3β(pSer9), and PKG2, with β-actin serving as a loading control. (C-E) Blots were obtained as in panel B, with independent POB isolates from three mice per genotype; they were analyzed by densitometry scanning using ImageJ. (F,G) Nuclear localization of β-catenin was determined by immuno¬fluo¬res¬cence staining of POBs isolated from three female mice per genotype. Some cells were treated with 100 µM 8-CPT-cGMP (+ cGMP) for 1 h (F). Some cells were pre-treated for 1 h with 4 mM N(ω)-nitro-l-arginine methyl ester (L-NAME) to inhibit NO production, and then were transferred to fresh medium with 4 mM L-NAME and incubated overnight prior to staining for β-catenin (G). Graphs show means ± SEM, *p/#p<0.05, **p/##p<0.01, and ***p/###p<0.001 for the indicated comparisons.
Supplemental Figure 3: Differentiation of POBs and BMSCs isolated from 8 week-old male Col1a1-prkg2RQ transgenic and wild type mice. POBs and BMSCs were isolated from eight week-old male transgenic (TG, n=5) mice and their wild type (WT, n=4) litter mates. (A, B) POBs were plated at high density and switched to differentiation medium after reaching confluency. After 14 d, cells were stained for ALP activity by staining with a colorimetric substrate (A). Mineralization was assessed after 21 d by Alizarin Red staining (B). Staining intensity was quantified by densitometry scanning. (C-E) Bone marrow mononuclear cells were plated at 4 x 105 cells/cm2, and adherent BMSCs were switched to osteoblastic differentiation medium for 14 d. BMSC colonies were assessed for mineralization by Alizarin Red staining (C). The relative mRNA abundance of osteocalcin (bglap), runx2, and actb (D) or prkg2 (E) was quantified by qRT-PCR and normalized to three housekeeping genes, as described in Fig. 2I. Data were calculated according to the ∆∆Ct method, with the mean of the WT group for each gene assigned a value of one. Data represent means ± SEM; *p<0.05 and **p<0.01 for comparison to WT.
Supplemental Figure 4: Micro-CT analysis of cortical parameters of 8 week-old male and female mice: Comparison of Col1a1-prkg2RQ transgenic and wild type littermates. In eight week-old male (A) and female (B) mice cortical bone area fraction, cross-sectional thickness and tissue mineral density (TMD) were measured by micro-CT at the mid-tibia as described in Methods. Data represent means ± SD (males: n=8 WT and n=10 TG; females: n=11 WT and n=10 TG).
Supplemental Figure 5: Calceine labeling of cortical bone in 8 week-old male mice and expression of Wnt-/osteoblast-, and RANKL-/osteoclast-related genes in tibiae from 8 week-old female mice. (A) Mineralizing surfaces (MS/BS), mineral apposition rates (MAR), and bone formation rates (BFR) were measured at endocortical surfaces between 5 and 0.25 mm proximal to the femoral growth plate in 8 week-old male Col1a1-prkg2RQ transgenic mice and wild type littermates. Data show the means ± SD of n=4 WT and n=5 TG mice; *p<0.05 and **p<0.01. (B,C) RNA was extracted from tibial shafts of 8 week-old female mice, and the relative abundance of (B) Wnt-/osteoblast- and (C) RANKL-/osteoclast-related transcripts was quantified by qRT-PCR and normalized to three different housekeeping genes (18S, hprt, and b2m). The mean of the WT group for each gene was assigned a value of one, as described in Fig. 5F. Gene names: Ctnnb1 (β-catenin), bglap (osteocalcin), alpl (alkaline phospha¬tase), spp1 (osteopontin), ccnd1 (cyclin D1), actb (β-actin), tnfsf11 (RANKL), tnfrsf11b (osteoprotegerin), acp5 (tartrate-resistant acid phosphatase) and ctsk (cathepsin K). Data represent means ± SEM from n=7 wild type and n=10 transgenic females per genotype. *p<0.05 for the comparison between WT and TG mice.
Supplemental Table 1: Primers Used for Quantitative RT-PCR