Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts

Abstract

Introduction: Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts.

Methods and results: We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts.

Conclusions: We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and suggest that gingival fibroblasts may have an ECM-degrading phenotype during conditions of hyperleptinaemia (e.g., obesity, type 2 diabetes mellitus, exogenous leptin therapy).

Fig 1. HGF morphology and vimentin expression.

(A) Light microscopy (100x magnification) of viable HGFs at high confluency displaying characteristic fibroblast morphology. HGFs stained with (B) no antibody control (C) anti-vimentin Ab. Images presented are from one donor and are representative of similar micrographs form three donors stained independently.

Fig 2. Effect of leptin on MMP mRNA and protein synthesis in HGFs.

HGFs were stimulated with leptin (0–25 μg/ml) for 24 h; MMP-1 gene expression was assessed by real-time RT-PCR (A) and supernatant MMP-1 concentrations were assessed by ELISA (B). HGFs were stimulated with leptin (10 μg/ml) for 24 h or the indicated durations and supernatant MMP-1 (C) and MMP-3 (D) concentrations were assessed by ELISA. HGFs were stimulated with leptin (0–25 μg/ml) for 24 h and MMP-14 gene expression was assessed by real-time RT-PCR (E). HGFs were stimulated with IL-1α (0.05 ng/ml) or OSM (5 ng/ml) alone, or in combination with leptin (10 μg/ml) (F) and pam2CSK4 (50 ng/ml) or E. coli LPS (10 ng/ml) alone, or in combination with leptin (10 μg/ml) for 24 h (G) and MMP-14 gene expression was determined by real-time RT-PCR. RT-PCR data (fold control) are expressed relative to RNAP. Data (excluding Fig 2D) are presented as mean+SEM HGF cultures from 3 HGF donors stimulated in independent experiments (n = 4 from each donor). Remaining data (Fig 2D) are expressed as mean+SD from 3 individual HGF donors (n = 4 from each donor). *P<0.05, **P<0.01, ***P<0.001 compared to the unstimulated control at the same time point.

Fig 3. Effect of IL-1 and OSM on MMP-1 and MMP-3 production by HGFs.

HGFs were stimulated with IL-1α (0.05 ng/ml), OSM (5 ng/ml) or IL-1α+OSM for 24 h. (A) MMP-1 gene expression was assessed by real-time RT-PCR and data (fold control) are expressed relative to RNAP. Supernatant MMP-1 (B) and MMP-3 (C) concentrations were assessed by ELISA. Data are presented as mean+SEM of three donors stimulated in independent experiments (n = 4 for each donor). **P<0.01, ***P<0.001 compared to the unstimulated control at the same time point.

Fig 4. Effect of leptin and IL-1, pam2CSK4 or LPS on MMP-1 and MMP-3 production by HGFs.

HGFs were stimulated with leptin (10 μg/ml), IL-1α (0.05 ng/ml) or leptin+IL-1α for 24 h (A-D). HGFs were stimulated with leptin (10 μg/ml), pam2CSK4 (50 ng/ml), or leptin+pam2CSK4 for 24 h (E-G). Inset (G): MMP-3 concentrations in HGFs isolated from donor 3 presented using an amplified scale. HGFs were stimulated with leptin (10 μg/ml), LPS (10 ng/ml) or leptin+LPS for 24 h (H). MMP-1 gene expression was assessed by real-time RT-PCR and data (fold control) are expressed relative to RNAP with the exception of Fig 4C which illustrates MMP-3 and 18S rRNA gene expression as assessed by RT-PCR and visualized on agarose gels. Supernatant MMP-1 concentrations were assessed by ELISA. Data (A, B, D, E) are presented as mean+SEM from 3 HGF donors stimulated in independent experiments (n = 4 from each donor). Remaining data (F, G, H) are expressed as mean+SD from 3 individual HGF donors (n = 4 from each donor). *P<0.05, ***P<0.001 compared to the unstimulated control at the same time point.

Fig 5. Surface TLR expression on HGFs.

HGFs were prepared for flow cytometry. (A) The region highlighted in this dot plot was used to gate cells and omit cellular debris. (B) Unstimulated HGFs were analysed for cell surface TLR2 and TLR4 expression by flow cytometry. These histograms are representative of results from 4 different HGF donors.

Fig 6. Investigation of leptin receptor expression and signalling pathways leading to MMP-1 synthesis in HGFs.

Flow cytometry for cell surface leptin receptor expression on unstimulated HGFs (A). Data are representative of similar data from 4 HGF donors. Inset (A): long LEPR isoform (Ob-Rb) and β2m gene expression as assessed by RT-PCR in unstimulated HGFs. Data are representative of similar results from 7 HGF donors. HGFs were stimulated with leptin (10 μg/ml), IL-1 (0.05 ng/ml), pam2CSK4 (50 ng/ml), OSM (5 ng/ml), leptin+IL-1 or leptin+pam2CSK4 for 20 min and lysates immunoblotted with the indicated antibodies including GAPDH as a loading control (B). The blots presented are all derived from the same donor and are representative of similar results from 3 donors stimulated in independent experiments. (C) HGFs were pre-treated with the ERK inhibitor U0126 (7.5 μM), (D) the JNK inhibitor SP600126 (10 μM), (E) STAT3 inhibitor VI (100 μM), or (F) the p38 inhibitor SB203580 (10 μM) for 30 min and then stimulated with leptin (10 μg/ml), IL-1α (0.05 ng/ml), pam2CSK4 (50 ng/ml), leptin+IL-1α or leptin+pam2CSK4 for 24 h. MMP-1 gene expression was assessed by real-time RT-PCR. Data (fold unstimulated DMSO-pre-treated control) are expressed relative to RNAP. Data are presented as mean+SEM of three donors stimulated in independent experiments (n = 4 for each donor). *P<0.05, **P<0.01, ***P<0.001 compared to the unstimulated control at the same time point.

Fig 7. Confirmation of the microarray dataset by real-time RT-PCR.

HGFs were stimulated with leptin (10 μg/ml), IL-1β (0.05 ng/ml) or leptin+IL-1β for 24 h. Real-time RT-PCR was used to assess (A) MMP-3, (B) MMP-8, (C) MMP-12, (D) collagen 6A3 (COL6A3), (E) MMP-14, and (F) MMP-2 gene expression. Data are expressed relative to RNAP and are presented as mean+SEM from three donors stimulated in independent experiments (n = 4 for each donor). *P<0.05, **P<0.01, ***P<0.001 compared to the unstimulated control at the same time point.