The role of leptin on the organization and expression of cytoskeleton elements in nucleus pulposus cells

Abstract

Obesity is an important risk factor for intervertebral disc degeneration and leptin is a biomarker of obesity. However, the expression of leptin receptors has not been determined in disc tissue. It is not known whether leptin has a direct effect on the nucleus pulposus (NP) cells. To determine whether the NP tissues and cells express leptin receptors (OBRa and OBRb) and whether leptin affects the organization and the expression of major cytoskeletal elements in NP cells. Messenger RNA (mRNA) and protein levels of OBRa and OBRb were measured by real-time PCR and Western blot, respectively, in NP tissues and cells. Immunofluorescence and real-time PCR and Western blot were performed to investigate the effect of leptin on cytoskeleton reorganization and expression. Results show that mRNA and proteins of OBRa and OBRb were expressed in all NP tissues and cells, and that OBRb expression was correlated with patients' body weight. Increased expression of β-actin and reorganization of F-actin were evident in leptin-stimulated NP cells. Leptin also induced vimentin expression but had no effect on β-tubulin in NP cells. These findings provide novel evidence supporting the possible involvement of leptin in the pathogenesis of intervertebral disc degeneration.

Figure 1

OBRa and OBRb expression in human NP cells. (A, B) Real-time RT-PCR analysis of OBRa and OBRb mRNAs in the human NP cells of eight patients. (C, D) Western blotting analysis of OBRa and OBRb protein expression in the human NP cells of eight patients. The signal in each lane was quantified using ImageJ software and the ratio of OBRs to GAPDH was determined.

Figure 2

The correlation between the expression of OBRa and OBRb and patients' BMI. (A) The correlation between the expression of OBRa and OBRb and patients' BMI. (B) Real-time RT-PCR analysis of OBRa and OBRb mRNAs in the human NP tissue of low-BMI group (BMI = 19.3, 19.5, 22.6, and 22.2) and high-BMI group (BMI = 31.6, 31.3, 30.25, and 28.04). (C) Western blotting analysis of OBRs protein expression in the human NP tissue of low-BMI group (BMI = 19.3, 19.5, 22.6, 22.2) and high-BMI group (BMI = 31.6, 31.3, 30.25, 28.04). Data are present as mean ± SD (n = 3). Error bars represent SD. *p < 0.05, when the low-BMI group compared to high-BMI group.

Figure 3

The effect of leptin on the F-actin cytoskeleton of human NP cells. (A) Fluorescence microscopy images showing arrangement of rhodamine–phalloidin-stained (red) F-actin filaments in primary human NP cells treated without or with leptin. Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40× objective. Control NP cells exhibited diffuses cytoplasmic and perinuclear staining of F-actin while leptin-stimulated NP cells showed strong cytoplasmic filamentous structures. Images are representative of three separate experiments. (B) The effect of leptin on β-actin mRNA expression assessed using real-time RT-PCR. For the dose-dependent studies NP cells were treated with either medium only or varying concentrations of leptin (1–1,000 ng/ml) for 24 h. For the time-dependent studies, NP cells were treated with either medium only or leptin (10 ng/ml) for varying time intervals (0–96 h). (C) The effect of leptin on β-actin protein expression assessed using Western blotting. For the dose-dependent studies quiescent NP cells were treated with either medium only or varying concentrations of leptin (1–1,000 ng/ml) for 24 h. For the time-dependent studies, quiescent NP cells were treated with either medium only or leptin (10 ng/ml) for varying time intervals (0–96 h).

Figure 4

The effect of leptin on the vimentin cytoskeleton of human NP cells. (A) Fluorescence microscopy images showing arrangement of vimentin cytoskeleton (blue) in primary human NP cells treated without or with leptin. Nuclei were stained with DAPI, shown in blue. Leptin did not appear to alter vimentin filament organization, although slightly stronger labeling was observed in the leptin-induced group compared to the control group. (B) The effect of leptin on vimentin mRNA expression assessed using real-time RT-PCR. For the dose-dependent studies quiescent NP cells were treated with either medium only or varying concentrations of leptin (1–1,000 ng/ml) for 24 h. For the time-dependent studies, NP cells were treated with either medium only or leptin (10 ng/ml) for varying time intervals (0–96 h). (C) The effect of leptin on vimentin protein expression assessed using Western blotting. For the dose-dependent studies NP cells were treated with either medium only or varying concentrations of leptin (1–1,000 ng/ml) for 24 h. For the time-dependent studies, quiescent NP cells were treated with either medium only or leptin (10 ng/ml) for varying time intervals (0–96 h).

Figure 5

The effect of leptin on the β-tubulin cytoskeleton of human NP cells. (A) Fluorescence microscopy images showing arrangement of β-tubulin cytoskeleton (blue) in primary human NP cells treated without or with leptin. Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40× objective. Leptin did not appear to alter β-tubulin filament organization in the leptin-induced group compared to the control group. (B) The effect of leptin on β-tubulin mRNA expression assessed using real-time RT-PCR. For the dose-dependent studies quiescent NP cells were treated with either medium only or varying concentrations of leptin (1–1,000 ng/ml) for 24 h. For the time-dependent studies, quiescent NP cells were treated with either medium only or leptin (10 ng/ml) for varying time intervals (0–96 h). (C) The effect of leptin on β-tubulin protein expression assessed using Western blotting. For the dose-dependent studies quiescent NP cells were treated with either medium only or varying concentrations of leptin (1–1,000 ng/ml) for 24 h. For the time-dependent studies, quiescent NP cells were treated with either medium only or leptin (10 ng/ml) for varying time intervals (0–96 h).

Figure 6

OBRa and OBRb expression in human NP tissue. Real-time RT-PCR analysis of OBRa (A) and OBRb (B) mRNAs in the human NP tissue of 37 patients. Real-time RT-PCR analysis was performed in triplicate and the expression levels of OBRa OBRb mRNAs were normalized to GAPDH mRNAs. Error bars represent standard derivation. (C) Western blotting analysis of OBRa and OBRb protein expression in the human NP tissue of eight patients. The signal in each lane was quantified using Image software and the ratio of OBRa and OBRb to GAPDH was determined.