Hepatocyte Growth Factor Regulates Macrophage Transition to the M2 Phenotype and Promotes Murine Skeletal Muscle Regeneration

Abstract

Hepatocyte growth factor (HGF) is well known for its role in the migration of embryonic muscle progenitors and the activation of adult muscle stem cells, yet its functions during the adult muscle regeneration process remain to be elucidated. In this study, we showed that HGF/c-met signaling was activated during muscle regeneration, and that among various infiltrated cells, the macrophage is the major cell type affected by HGF. Pharmacological inhibition of the c-met receptor by PHA-665752 increased the expression levels of pro-inflammatory (M1) macrophage markers such as IL-1β and iNOS while lowering those of pro-regenerative (M2) macrophage markers like IL-10 and TGF-β, resulting in compromised muscle repair. In Raw 264.7 cells, HGF decreased the RNA level of LPS-induced TNF-α, IL-1β, and iNOS while enhancing that of IL-10. HGF was also shown to increase the phosphorylation of AMPKα through CaMKKβ, thereby overcoming the effects of the LPS-induced deactivation of AMPKα. Transfection with specific siRNA to AMPKα diminished the effects of HGF on the LPS-induced gene expressions of M1 and M2 markers. Exogenous delivery of HGF through intramuscular injection of the HGF-expressing plasmid vector promoted the transition to M2 macrophage and facilitated muscle regeneration. Taken together, our findings suggested that HGF/c-met might play an important role in the transition of the macrophage during muscle repair, indicating the potential use of HGF as a basis for developing therapeutics for muscle degenerative diseases.

Keywords: AMPK; CaMKKβ; hepatocyte growth factor; macrophage transition; muscle regeneration.

Figure 1

HGF/c-met signaling is activated during muscle regeneration. (A) Expression kinetics of HGF protein during muscle injury by CTX and regeneration. The muscle was isolated at 2, 4, 7, and 12 days after CTX injection, and total proteins were analyzed by ELISA to measure the protein levels of HGF. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 versus sham-treated muscles (unpaired student’s t test), n = 4 per group. (B) Expression kinetics of the RNA levels of HGF during muscle injury and regeneration. RNA was prepared from TAs 1, 3, and 7 days after CTX injection followed by RT-qPCR, ^*p < 0.05, ^**p < 0.01 versus sham-treated muscles (unpaired student’s t test), n = 4 per group. The values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (C) Expression kinetics of total and phosphorylated c-met proteins in CTX-injured TAs. Muscles were isolated at 3 and 7 days postinjury, and total proteins were prepared followed by western blot using specific antibodies to total or phosphorylated c-met. β-tubulin was used as a loading control. Each lane represents a sample from an individual mouse. Two representative mice are shown here. Two independent experiments were performed (with a total of four mice), and similar results were obtained. Arrow indicates the protein of interest in blots. (D) Identification of cell types expressing c-met. CTX-injured TA was isolated 3 days postinjury and subjected to immunofluorescence assay using antibodies to CD11b for macrophages, CD31 for endothelial cells, Ly6G for neutrophils (all red), and phosphorylated c-met (green). Nuclei were counterstained with DAPI (blue). n = 4 per group. Scale bars, 20 μm. All data are presented as mean ± SEM. See also Supplementary Figure S1.

Figure 2

Effects of c-met inhibitor PHA-665752 on muscle regeneration. After CTX injury, mice were i.p. injected with 20 mg/kg of PHA-665752 on a daily basis until sacrificed. CTX injured TAs were analyzed 7 days postinjury. (A) Effect on muscle weight. Muscle mass was normalized with the weight of mice. PHA, PHA-665752. ns, not significant, ^**p < 0.01, ^***p < 0.001 (one-way ANOVA), n = 4 per group. (B) H&E staining of regenerating muscle. Crosses (x) and asterisks (*) indicate necrotic and phagocyted myofibers, respectively. Scale bars, 200 μm. (C) Effect on cross-sectional areas of muscle fibers. Mean value of area sizes is indicated in the graph. ^**p < 0.01, ^***p < 0.001 (one-way ANOVA), n = 4 per group. (D) Quantification of necrotic or phagocyted fibers expressed as a percentage of total myofibers. At least 300 muscle fiber areas were counted per sample. ^**p < 0.01, ^***p < 0.001 (unpaired student’s t test), n = 4 per group. All data are presented as mean ± SEM. See also Supplementary Figure S2.

Figure 3

Effects of c-met inhibitor PHA-665752 on macrophage population infiltrated in the muscle. After CTX injury, mice were i.p. injected with 20 mg/kg of PHA-665752 on a daily basis until sacrificed. (A) Effects on the RNA levels of M1 markers (iNOS, IL-1β, and CCL2). (B) Effects on the RNA levels of M2 markers (IL-10, TGF-β, Arg1, CD206, CD163, Ym1, and Retnla). The RNA level of these genes isolated from TAs 3 days after injury was determined by RT-qPCR. The relative expression level of sham-operated, vehicle-treated mice is presented as 1. (C) Effect on iNOS-positive macrophages and (D) CD206-positive macrophages. CTX-injured TA was isolated 3 days postinjury and subjected to immunofluorescence assay using antibodies to CD11b (red) and iNOS or CD206 (green). Nuclei were counterstained with DAPI (blue). Percentage of iNOS+ or CD206+ macrophages was indicated in the graph. ^*p < 0.05, ^**p < 0.01 (unpaired student’s t test), n = 4 per group. Scale bars, 20 μm. (E) Effects on MyoD RNA. ^*p < 0.05 (unpaired student’s t test), n = 4 per group. (F) Effects on MyoD protein. Two representative results are shown here, n = 4 per group. All data are presented as mean ± SEM.

Figure 4

Roles of CaMKKβ-AMPKα on HGF-mediated control of the expression of M1 and M2 markers in Raw 264.7 cells. Raw 264.7 cells were cultured in the presence or absence of LPS and recombinant HGF proteins. Total RNA and proteins were prepared and analyzed by RT-qPCR and western blot, respectively. (A) Effects of HGF on the RNA levels of M1 markers (IL-1β, iNOS, and TNFα). ^*p < 0.05, ^**p < 0.01 (unpaired student’s t test), n = 3 per group. (B) Effects of HGF on RNA levels of M2 markers (IL-10 and Arg1). Values were normalized to GAPDH. ^*p < 0.05, ^**p < 0.01 (unpaired student’s t test), n = 3 per group. (C) Effects of HGF on signaling pathways related to macrophage polarization. (D) Effects of AMPKα knockdown on HGF-mediated regulation of the RNA level of IL-1β, iNOS, and IL-10, marker genes of M1 and M2. Raw 264.7 cells were transfected with AMPKα or control siRNAs, and then treated with LPS and HGF. Values were normalized to GAPDH. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (unpaired student’s t test), n = 3 per group. (E) Effects of CaMKKβ inhibitor, STO-609, on the HGF-mediated phosphorylation of AMPKα. Arrow indicates the protein of interest in blots. In western blot hybridization, GAPDH was used as a loading control. All data are presented as mean ± SEM. See also Supplementary Figure S2 and S3.

Figure 5

Effects of HGF overexpression by intramuscular injection of HGF expressing plasmid on muscle regeneration. pCK or pCK-HGF-X7 was i.m. injected 3 days prior to the CTX injection. TAs were prepared at appropriate times after injury. (A) Effect on TA weight. Representative TAs from 7 days postinjury are shown in the photos. ^*p < 0.05 versus CTX + pCK group (one-way ANOVA), n = 4 per group. Scale bar, 1 mm. (B) Effects on cross-sectional areas of regenerating fibers. CTX-injured TA was isolated 3 days postinjury and subjected to immunofluorescence assay using antibodies to eMHC (red) for regenerating myofibers. Percentage of eMHC+ myocytes was indicated in the graph. ^***p < 0.001 (one-way ANOVA), n = 4 per group. Scale bar, 50 μm. (C–E) Three days after injury, the TA was isolated, and total RNAs were analyzed by RT-qPCR. (C) Effects on the expression of Myh3. ^*p < 0.05 (one-way ANOVA), n = 4 per group. (D) Effects on the RNA levels of M1 markers (IL-1β, iNOS, and CCL2). ^*p < 0.05 (one-way ANOVA), n = 4 per group. (E) Effects on RNA levels of M2 markers (IL-10, TGF-β, Arg1, and CD163). ^*p < 0.05 (one-way ANOVA), n = 4 per group. Values were normalized to GAPDH. All data are presented as mean ± SEM. See also Supplementary Figure S4.