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 4

Reduced Vegfa and Vegfr1 expression in PKG1-deficient osteoblasts and bones and decreased capillary density in regenerating bone of Prkg1 OB-KO mice.

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Reduced Vegfa and Vegfr1 expression in PKG1-deficient osteoblasts and bo...
(A) Transcripts of vascular endothelial growth factor A (Vegfa), and its receptor (Vegfr1), were quantified by qRT-PCR together with Hprt mRNA in tibiae of Prkg1 OB-KO mice (Col1a1CRETg/+ Prkg1fl/fl) and control littermates (Prkg1fl/fl); mRNA amounts were normalized to 18S rRNA, and the mean mRNA level found in control tibiae was assigned a value of 1 (n = 7 mice per genotype). (B) Vegfa, Vegfr1, and Hprt mRNAs were measured in POBs isolated from Prkg1 OB-KO and control mice. Values were normalized to 18S rRNA, and the mean mRNA level found in control cells was assigned a value of 1 (n = 5 independent experiments). (C and D) 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 6 or 24 hours, as indicated. (C) Vegfa and (D) Vegfr1 mRNAs were quantified and normalized to the mean level of vehicle-treated control cells as described in panel B (n = 3 independent experiments for 6 hours and n = 5 for 0- and 24-hour time points). (E) Prkg1 OB-KO mice and control littermates were subjected to drill hole surgery as described in Figure 2. Immunostaining was performed with anti-CD31 antibody (or control IgG) on longitudinal tibial sections through the drill-generated defect; shown is the 0.1-mm2 “region of interest,” defined as in Figure 2C. Capillary density was assessed based on single CD31-immunoreactive endothelial cells and endothelial cell clusters separate from other microvessels (red arrowheads); the graph represents n = 5 mice/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 (E), or 2-way ANOVA (C and D); ##P < 0.01, and ###P < 0.001 for the comparison with control cells under the same treatment condition.