Protein kinase G1 regulates bone regeneration and rescues diabetic fracture healing
Nadine Schall, Julian J. Garcia, Hema Kalyanaraman, Shyamsundar Pal China, Jenna J. Lee, Robert L. Sah, Alexander Pfeifer, Renate B. Pilz
Nadine Schall, Julian J. Garcia, Hema Kalyanaraman, Shyamsundar Pal China, Jenna J. Lee, Robert L. Sah, Alexander Pfeifer, Renate B. Pilz
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Research Article Bone biology

Protein kinase G1 regulates bone regeneration and rescues diabetic fracture healing

Abstract

Bone fractures are a major cause of morbidity and mortality, particularly in patients with diabetes, who have a high incidence of fractures and exhibit poor fracture healing. Coordinated expression of osteoblast-derived vascular endothelial growth factor (VEGF) and bone morphogenic proteins (BMPs) is essential for fracture repair. The NO/cGMP/protein kinase G (PKG) signaling pathway mediates osteoblast responses to estrogens and mechanical stimulation, but the pathway’s role in bone regeneration is unknown. Here, we used a mouse cortical-defect model to simulate bone fractures and studied osteoblast-specific PKG1-knockout and diabetic mice. The knockout mice had normal bone microarchitecture but after injury exhibited poor bone regeneration, with decreased osteoblasts, collagen deposition, and microvessels in the bone defect area. Primary osteoblasts and tibiae from the knockout mice expressed low amounts of Vegfa and Bmp2/4 mRNAs, and PKG1 was required for cGMP-stimulated expression of these genes. Diabetic mice also demonstrated low Vegfa and Bmp2/4 expression in bone and impaired bone regeneration after injury; notably, the cGMP-elevating agent cinaciguat restored Vegfa and BMP2/4 expression and full bone healing. We conclude that PKG1 is a key orchestrator of VEGF and BMP signaling during bone regeneration and propose pharmacological PKG activation as a novel therapeutic approach to enhance fracture healing.

Authors

Nadine Schall, Julian J. Garcia, Hema Kalyanaraman, Shyamsundar Pal China, Jenna J. Lee, Robert L. Sah, Alexander Pfeifer, Renate B. Pilz

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Figure 5

Impaired BMP2/4 expression and BMP signaling in POBs and bones from Prkg1 OB-KO mice.

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Impaired BMP2/4 expression and BMP signaling in POBs and bones from Prkg...
(A) Transcripts of BMPs (Bmp2 and Bmp4) and their receptors (Bmpr1a and Bmpr2) were quantified by qRT-PCR in tibiae of Prkg1 OB-KO mice (Col1a1CRETg/+ Prkg1fl/fl) and control littermates (Prkg1fl/fl); mRNA levels were normalized as in Figure 4A (n = 7 mice per genotype). (B) BMP-related transcripts were measured in POBs isolated from Prkg1 OB-KO and control mice. Values were normalized as described in Figure 4B (n = 6 independent experiments). (C) POBs from control and Prkg1 OB-KO mice were cultured in medium containing 0.5% FBS for 24 hours before receiving 100 μM 8-CPT-cGMP (gray bars) for 24 hours. Bmp2, Bmp4, Bmpr1, and Bmpr2 mRNAs were quantified and normalized as in Figure 4B, with levels in vehicle-treated control cells assigned a value of 1 (n = 3 independent experiments for 6 hours and n = 5 for 0- and 24-hour time points). (D) POBs from control and Prkg1 OB-KO mice were cultured as in panel C but received vehicle or 1 nM BMP-2 for the indicated times, and Smad-1/5/8 phosphorylation was assessed by Western blotting using Smad-pSer463/465–specific antibodies. Blots were quantified by ImageJ and normalized to β-actin (n = 4 independent experiments). (E) Prkg1 OB-KO mice and control littermates were subjected to drill hole surgery as described in Figure 2. Immunostaining was performed with phospho-Smad1/5/8 (pSer463/465) antibody (or control IgG) on longitudinal tibial sections through the drill-generated defect. Phospho-Smad1/5/8–positive brown nuclei (red arrows show examples, scale bars: 25 μm) were counted in the 0.1-mm2 “region of interest” (defined in Figure 2C). The graph summarizes results for n = 5 Prkg1 mice per genotype. Graphs show means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 for comparison by 1-sample t test (A and B), 2-sided t test (F), or 2-way ANOVA (C and D).