cGMP-dependent protein kinase-2 regulates bone mass and prevents diabetic bone loss

Abstract

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 (PKG2) 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- 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 PKG2 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.

Protein kinase G1 regulates bone regeneration and rescues diabetic fracture healing

“…PKG1-deficient and control osteoblasts proliferated similarly when stimulated with the membrane-permeable cGMP analog 8-CPT-cGMP or with 20% fetal bovine serum, measured after a period of growth factor deprivation in medium containing 0.1% FBS ( Figure 3C). These data are consistent with PKG2, rather than PKG1, mediating proliferative responses of osteoblasts to various growth stimuli, including cGMP (18,21,26).…”

“…We crossed mice carrying Prkg1 alleles flanked by loxP sites (Prkg1 fl/fl ) with mice expressing Cre recombinase under control of the 2.3-kb collagen1a1 promoter (Col1a1-CRE Tg/+ ) -both in a C57BL/6 background -to generate mice with osteoblast-specific Prkg1 knockout (genotype Col1a1-CRE Tg/+ Prkg1 fl/fl ), as described in Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.135355DS1. We and others have shown that the 2.3-kb Col1a1 promoter is specifically expressed in cells of osteoblastic lineage, from committed osteoblast precursors to mature osteocytes (21,22). Prkg1 mRNA in tibial bone shafts was reduced by more than 80% in transgene-positive Prkg1 OB-KO mice compared with control, transgene-negative Prkg1 fl/fl littermates, while Prkg2 mRNA was not significantly reduced ( Figure 1A).…”

“…To minimize the influence of estrogens on NO/cGMP production (12,21), we studied male mice. We analyzed their bones ex vivo using 3D μCT scans 10 days after surgery (Supplemental Figure 3).…”

“…To induce diabetes, we administered streptozotocin to 10-week-old male C57BL/6 mice, using male mice again to minimize estrogen effects on NO/cGMP production (12,21). Drill hole surgery was performed 4 weeks later (after mice had been hyperglycemic for at least 2 weeks), and on postoperative day 1, we started daily cinaciguat injections until euthanasia on day 10 ( Figure 6A).…”

The NO–cGMP–PKG pathway in skeletal remodeling

“…Primary osteoblasts from Prkg2 –/– mice showed decreased c‐fos (Fos) and Fra 2 ( Fosl2 ) expression, which are involved in osteoblast cell cycle regulation 34 . On the other hand, osteoblast‐specific PKG2 gain‐of‐function mutation (Col1a1‐ Prkg2 RQ ) showed significantly increased bone formation parameters, such as trabecular mineralizing surface, MAR, and BFR compared with littermates 37 . Interestingly, this skeletal phenotype was more evident in male mice, suggesting the possible interaction of sex hormones and the NO–cGMP–PKG2 signal.…”