GIRK3 deletion facilitates kappa opioid signaling in chondrocytes, delays vascularization and promotes bone lengthening in mice

Abstract

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K⁺ (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2^-/-, Girk3^-/- and Girk2/3^-/- mice. Bones from 12-week-old Girk2^-/- mice were normal in length, but femurs and tibiae from Girk3^-/- and Girk2/3^-/- mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3^-/- mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3^-/- chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3^-/- mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

Keywords: Cartilage; Development; G protein; GPCR; K(+) channel; Kappa opioid.

Figure 1.. Girk3^−/− mice have longer femora and tibiae than WT mice.

(A) Femur lengths of male and female WT, Girk2^−/−, Girk3^−/−, and Girk2/3^−/− mice were measured at 12 weeks of age following dissection and x-ray. (B) X-rays of WT and Girk2/3^−/− male mice at 12 weeks of age. Yellow dashed lines indicate the WT bone length. Scale bar indicates 5 mm. (C) Femur lengths of male and female WT and Girk2^−/− mice were measured at 12 weeks of age following dissection and x-ray. Statistics were performed using Student’s T-test. (D) X-rays of WT and Girk2^−/− female mice at 12 weeks of age. Yellow dashed lines indicate the WT bone length. Scale bar indicates 2.5 mm. (E) X-rays of WT and Girk3^−/− male mice at 4 weeks and 12 weeks of age. Yellow dashed lines indicate the WT bone length at each respective age. Scale bar indicates 5 mm. (F) Post-weaning growth rates of male femurs and tibiae from WT and Girk3^−/− male and female mice. Bone lengths were determined using calipers. Statistics were performed using a two-way ANOVA with repeated measures. * : P<0.05, ** : P<0.01

Figure 2.. Girk3 deletion alters the chondro-transcriptome.

(A) Heat maps of differentially regulated genes in Girk3^−/− versus WT chondrocytes. RNA was collected from freshly isolated epiphyseal chondrocytes and prepared for bulk RNA-Seq. Semi-quantitative PCR of (B) Girk3, (C) Col2a1, Col4a6, Col10a1, Sox9, (D) Oprk1, Pdyn, (E) Kdr, and Vegfa was performed on freshly isolated chondrocytes from independent populations of WT and Girk3^−/− mice. Statistics were performed using Student’s T-test.

Figure 3.. Girk3 deletion increases kappa opioid-induced extracellular matrix production in chondrocyte cultures.

(A) GIRK3 and Actin were measured in cell lysates prepared from freshly isolated epiphyseal chondrocytes of WT and Girk3^−/− mice via Western blotting. (B) Live cell imaging was performed over 48 hours on WT and Girk3^−/− IMCs plated in monolayer and cell proliferation was determined based off cell confluency. (C) Alcian Blue staining showing matrix production by WT and Girk3^−/− micromasses in response to Dynorphin (Dyn; 0.1μM) with or without Norbinaltorphimine (Nor-B; 20μM) after 6 days. (D, E) Glycosaminoglycan (GAG) production was measured in micromasses after (C) 3 and (D) 6 days in the indicated conditions. Statistics were performed using a two-way ANOVA.

Figure 4.. Girk3 deletion increases kappa opioid induced CREB activation in chondrocyte cultures.

WT or Girk3^−/− IMCs were cultured in monolayer in the presence of vehicle, Dyn and/or Nor-B. (A) Cyclic AMP production in the supernatant was measured via ELISA 15 min after 1μM Dyn and/or 20μM Nor-B were added to the culture. (B) pCREB1 and total CREB1 production were measured in cell lysates 45 minutes after 1μM Dyn and/or 20μM Nor-B were added. (C, D) QPCR was performed to measure expression levels of (C) Col2a1 and (D) Sox9 after 6 days in culture with 0.1μM Dyn and/or 20μM Nor-B. Statistics were performed using a two-way ANOVA.

Figure 5.. Vascularization of the primary spongiosa is reduced in Girk3^−/− mice.

(A-B) RNAScope in situ hybridization was performed using probes targeted to Kdr and Flt1 on 1-week-old male WT and Girk3^−/− mice. Images were taken at 10x (scale bar = 50 μm) or 20x (scale bar = 100 μm) to visualize positive brown staining in the primary spongiosa. (C) Maximum intensity projection of confocal images showing Endomucin (green) and DNA (DAPI, blue) in 3-week-old male WT and Girk3^−/− mice. Scale bar indicates 100 μm. “gp” = growth plate. (D,E) RNAScope in situ hybridization was performed using a probe targeted to Mmp13 on 1-week-old male WT and Girk3^−/− mice. Images were taken at 5x (scale bar = 50 μm) or 20x (scale bar = 100 μm) to visualize positive brown staining. (E) Number of Mmp13-positive brown stained cells (Mmp13+) were quantified along the chondro-osseous border.