Serum p-AMPK-α1 levels positively correlated with disease activity in RA
To investigate the difference in AMPK levels between RA and OA patients and to detect the presence of correlation between AMPK levels and RA disease activity, we determined AMPK-α1 and p-AMPK-α1 levels using 20 OA and 61 RA patients having different disease activities. No significant differences exist in AMPK-α1 levels between OA and RA patients (Figure 1A). However, p-AMPK-α1 levels were higher in OA compared to RA patients, who had lower disease activity (p < 0.01) (figure 1B). This significance level was increased when the values for p-AMPK-α1 levels were log transformed (p < 0.0001). Interestingly, after log transformation, RA patients having higher disease activity was found to have significantly higher levels of p-AMPK-α1 compared to patients having low disease activity (p < 0.01) (Figure 1C). In addition, p-AMPK-α1 levels were positively correlating with DAS28 scores (r = 0.270, 95%CI: 0.142 - 0.492, p < 0.0001) and CRP levels (r = 0.259, 95%CI: 0.009 - 0.478, p < 0.05) (Figure 1D and E). However, such a correlation did not exist with ESR levels (Figure 1F).
IL-17 and TNF-α levels were reported to have correlation with RA activity and thus were currently selected as therapy targets (13, 14). We analyzed the presence of possible correlation between AMPK levels with IL-17 and TNF-α in the serum samples. The results demonstrated an increased expression of both the inflammatory cytokines in RA than OA patients, and AMPK-α1 levels were moderately correlated with TNF-α levels (r = 0.46, 95% CI: 0.241-0.640, p = 0.0002) (Figure 1G, H & I). However, no statistical correlation exists between log transformed p-AMPK-α1 and IL-17 or TNF-α levels (supplementary file 2).
Although AMPK is a key regulator of glucose metabolism (15), and RA and other autoimmune diseases appear to be associated with an increased risk of diabetes mellitus (16), no differences in glucose levels between groups were observed in our study (supplementary file 2).
AMPK levels were more significantly present in RA synovial samples
Since AMPK has an anti-inflammatory effect in many inflammation related diseases (17, 18), and our data showed a mild positive correlation with disease activity, we next evaluated the data available in NCBI GEO Profiles [accession numbers: GSE12021(11), GSE55235, and GSE55457(12)] for AMPK levels from RA and OA patients’ synovial tissue samples. In total, 119 DEGs intersected in all the three datasets (supplementary file 3), which demonstrated the high consensus existing between results from different experiments. Although AMPK was not directly detected in the analysis, one of the most obviously changed pathway was the metabolic pathway consisting of 9 genes (Figure 2A). Since AMPK has a controlling function in metabolism (6, 19), we focused on its expression at both protein and mRNA levels in the synovium samples from OA and RA patients. The immunohistochemical staining revealed higher level of p-AMPK-α1 expression in RA than OA synovium (Figure 2B). Similarly, relative expression levels of AMPK-α1, AMPK-α2 and AMPK-γ3 genes were higher in synovium of RA than OA patients (Figure 2C).
Metabolism variations in FLS
Phosophorylated AMPK-α1 expressed in the proliferating FLS identified using IHC staining was confirmed by immunofluorescence studies (see supplementary file 2). FLS are the most common cell types present at the pannus-cartilage junction, which contribute to joint destruction through their production of cytokines, chemokines and matrix-degrading molecules as well as by migrating and invading the joint cartilage (20), and thus regarded as key effector cells(21). Hence, we analyzed the differences in the expression of genes between RA and OA FLS by high-throughput RNA sequencing method. The results demonstrated that among the 111 DEGs identified, expression of 95 genes were up-regulated and 16 genes were down regulated in RA than OA FLS (Figure 3A and to see all the DEGs present in the individual samples refer supplementary file 4). KEGG pathway enrichment analysis suggested involvement of metabolic and glycerolipid metabolism pathways with 9 genes closely related to AMPK regulation (Figure 3A, B). Among them, diacylglycerol kinase gamma (DGKG) and prostaglandin D2 synthase (PTGDS) expressions were down regulated, while the expression of other 7 genes, lipase F (LIPG), heparanase (HPSE), glycerol-3-phosphate acyltransferase 2 (GPAT2), phospholipase A2 group VII (PLA2G7), choline dehydrogenase (CHDH), ST6 b-galactoside a-2,6-sialyltransferase 2 (ST6GAL2) and ST8 a-N-acetyl-neuraminide a-2,-sialyltransferase 5 (ST8SIA5) were up regulated in RA compared to OA FLS. Interestingly, hyaluronan and proteoglycan link protein 1 (HAPLN1) expression was highly up-regulated in RA-FLS (Figure 3A)
Metformin affects FLS proliferation
Based on our above results and prior knowledge on anti-inflammatory functions of AMPK (7, 8), we deduced that up-regulation of AMPK-α1 expression in RA synovium might be due to an inflammation stress. So, we used metformin (22) and dorsmorphin (23) as AMPK activator and inhibitor, respectively to evaluate their effects on FLS proliferation.
FLS were cultured with different concentrations of metformin or PBS in 6-well plates and observed between 0-72 hours under inverted microscope for their viability. MTT assay was performed to confirm the cellular viability of FLS after metformin treatment. Both the results demonstrated inhibition of FLS proliferation by metformin at 5 and 10 mM concentrations. However, at a low concentration (1 mM) metformin promoted FLS proliferation (Figure 4A, B), which was further confirmed by reducing the concentration further. Importantly, metformin inhibited FLS proliferation even at 2 mM concentration (Figure 4C), demonstrating dose-dependent effect of metformin on FLS proliferation. In contrast, dorsmorphin promoted FLS proliferation significantly at very low (5 and 10 μM) concentrations.
Metformin inhibited FLS migration
Based on the concentration gradient of metformin for its inhibitory effects on FLS proliferation, we selected metformin at 5 mM as well as dorsmorphin at 5 μM to study their effect on FLS migration ability by testing wound repair rate in a scratch test experiment. Interestingly, both metformin and dorsmorphin inhibited FLS migration significantly (Figure 5). Earlier, dorsmorphin was reported to inhibit the migration of certain cancer cells and this phenomenon was explained by AMPK-independent mechanisms (23).
Metformin increased AMPK-α1 and HAPLN1 expression
Results from semi-quantification of mRNA levels in FLS after metformin treatment by RT-qPCR showed a significant decrease in IL-6 gene expression, while the expressions of AMPK-α1, PKA-α and HAPLN1 genes were significantly increased (Figure 6A). Automated electrophoresis western blot analysis confirmed the up regulation of p-AMPK-α1 and HAPLN1 at the protein level (Figure 6H).
The role of IL-6 in the pathogenesis of joint and systemic inflammation in RA has been clearly demonstrated (24), and IL-6 inhibitor has been used for the treatment of RA with favorable outcomes (25). Our results confirmed AMPK-dependent effects of metformin on IL-6 gene expression (26, 27) as we noticed a significant negative correlation (r = -0.422, 95%CI: -0.672 to -0.0865, p = 0.016) between AMPK-α1 and IL-6 gene expressions (Figure 6B). PKA-α is a regulatory subunit of the cAMP-dependent protein kinases involved in cAMP mediated signaling events in the cells and a mutual promotion effect between AMPK and PKA-α had been reported earlier (28, 29). In this study, we found a significant increase in PKA-α gene expression in FLS after metformin treatment. AMPK also phosphorylates the mammalian target of rapamycin complex 1 (mTORC1) subunit, regulatory associated protein of mTOR (RAPTOR), which is essential for AMPK function as a metabolic checkpoint (30). Although RAPTOR and mTOR did not have any significant changes after metformin treatment, a negative correlation between RAPTOR and AMPK-α1 expression was detected (r = -0.470, 95%CI: -0.682 to -0.185, p = 0.002) (Figure 6C) confirming an earlier report (31). This pathway was reported to regulate cell growth in response to nutrient and insulin levels.
Interestingly, after treating FLS with metformin, an up-regulation of HAPLN1 expression was observed, which was significantly positive correlated with AMPK-α1 gene expression (r = 0.560, 95%CI: 0.308 to 0.738, p < 0.0001, Figure 6D) as well as at protein level (r = 0.785, 95%CI: 0.3869 to 1.238, p = 0.0015, Figure 6I). HAPLN1 was reported as one of the distinctive genes expressed in RA FLS correlating with the disease activity (32). However, effects of metformin on AMPK-α1 expression and subsequent modulation of HAPLN1 has not been reported earlier. HAPLN1 interacts with the globular domains of hyaluronic acid and proteoglycans to form stable ternary complexes in various extracellular matrices. Its main biological function is to maintain the stable aggregation of hyaluronic acid and proteoglycan monomers in the extracellular cartilage matrix (33), and to stabilize the binding interactions between hyaluronic acid and chondroitin sulfate, which contribute to the compression resistance of joints (34). Perinatal mice containing targeted mutations in the HAPLN1 gene developed lethal cartilage dysplasia (35) suggesting the essential role of HAPLN1 as a regulator of cartilage homeostasis and formation.
In granulosa cells, HAPLN1 was proposed to be promoted through PKA-RUNX1/RUNX2 pathway (36). Although RUNX1 and RUNX2 expressions in metformin treated FLS were not significantly changed, RUNX1 expression was negatively correlating with AMPK-α1 expression (r = -0.339, 95%CI: -0.604 to -0.007, p < 0.046), while having a positive correlation with HAPLN1 (r = 0.547, 95%CI: 0.291 to 0.729, p < 0.0001, Figure 6E and G) expression. Conversely, HAPLN1 expression did not show any significant correlation with RUNX2, though AMPK-α1 and RUNX2 expressions were positively correlated (r = 0.471, 95%CI: 0.103 to 0.656, p = 0.011, Figure 6F). However, our current study cannot explain this contradictory observation because of the prevailing extremely complicated molecular interactions in vivo. RUNX1 controls anergy and suppressive functions of regulatory T-cells by associating with FOXP3. It activates the expression of IL-2 and IFN-γ and down-regulates the expression of TNF receptor superfamily member 18 (TNFRSF18), IL-2 receptor subunit alpha (IL-2RA) and cytotoxic T-lymphocyte associated protein 4 (CTLA4) in conventional T-cells(37), while positively regulating the expression of RORC in T-helper 17 cells (38). On the other hand, RUNX2 is essential for the maturation of osteoblasts and has an important role in the intramembranous and endochondral ossification processes (39, 40). Based on our knowledge, we summarized possible mode of action of metformin on FLS in figure 5 of online supplementary file 2.