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 1

Reduced osteoblastic gene expression and bone formation rates in mice with osteoblast-specific Prkg1 deletion.

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Reduced osteoblastic gene expression and bone formation rates in mice wi...
(A) Prkg1 and Prkg2 mRNA were quantified by quantitative reverse-transcription PCR (qRT-PCR) in control (genotype Prkg1fl/fl) and osteoblast-specific Prkg1-KO mice (Prkg1 OB-KO, genotype Col1a1CRETg/+ Prkg1fl/fl); mRNA expression was normalized to 18S rRNA, and the mean relative mRNA level found in control mice was assigned a value of 1 (n = 7–8 mice per genotype). (B) Immunohistochemical staining with an antibody specific for PKG1 in tibial sections from control and Prkg1 OB-KO mice (arrows point to osteoblasts; representative for 3 mice per genotype). Staining of megakaryocytes (M) served as positive control and control IgG as negative control (scale bars: 25 μm). (C) Expression of osteoblast differentiation-related genes (alkaline phosphatase, Alpl; osteocalcin, Bglap; collagen-1α1, Col1a1) and the housekeeping gene hypoxanthine phosphoribosyltransferase (Hprt) was measured in tibial bone of control (black bars) and Prkg1 OB-KO mice (gray bars) and was normalized as described in panel A (n = 6 mice per genotype). (D) Osteoblasts were counted on trabecular surfaces of trichrome-stained tibial sections; values are expressed as number per millimeter of bone perimeter (n = 6 mice per genotype). (E and F) Control and Prkg1 OB-KO mice, 8 weeks old, were injected with calcein at 7 and 2 days before euthanasia, respectively, and trabecular labeling was assessed by fluorescence microscopy of tibial sections. Mineralizing surfaces (MS/BS), mineral apposition rates (MARs), and bone formation rates (BFRs) were measured on trabecular surfaces as described in Methods (n = 6 mice per genotype). (G and H) Expression of RANKL (Tnfsn11) and osteoprotegerin (Tnfrsf11b) and of the osteoclast-specific genes tartrate-resistant acid phosphatase (TRAP; Acp5) and cathepsin K (Ctsk) was measured by qRT-PCR in control (black bars) and Prkg1 OB-KO mice (gray bars) and normalized as described in panel A (n = 7–8 mice per genotype). (I) Osteoclasts were counted on trabecular surfaces of trichrome-stained tibial sections (n = 6 mice per genotype). Graphs show means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 by 1-sample t test (panels A, C, G) or 2-sided t test (F).