Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes

<div><p>Bone morphogenetic proteins (BMPs) play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. However, recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA) development. The aim of this study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT) 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs) 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptin-induced OA development.</p></div

Leptin induces BMP-2 expression in human primary chondrocytes.

<p>Cells were kept as controls or stimulated with leptin for 1, 4, 8, and 24 h, and the BMPs mRNA expressions (<i>A</i>) and protein secretion (<i>B</i>) were determined by real-time PCR and ELISA, respectively. Data in (<i>A</i>) and (<i>B</i>) are mean ± SEM from three independent experiments. *, <i>P</i> < 0.05 <i>vs</i>. untreated control cells.</p

Leptin-induced BMP-2 mRNA expression is mediated by JAK2-ERK1/2 signaling in human primary chondrocytes.

<p>(<i>A</i>) Cells were kept as controls or treated with leptin for 10’, 30’, 1, 4, and 8 h, and the phosphorylations of JAK1/2 and ERK1/2 were determined by Western blot. (<i>B</i>-<i>D</i>) Cells were transfected with (<i>B</i>-<i>C</i>) JAK1- or JAK2-specific siRNA or (<i>D</i>) ERK1- or ERK2-specific siRNA and then kept as controls or treated with leptin for 4 h. (<i>B</i>) The phosphorylation of ERK1/2 was determined by Western blot and (<i>C</i>-<i>D</i>) the BMP-2 mRNA expression was determined by real-time PCR. Results in (<i>A</i>) and (<i>B</i>) are representative of three independent experiments with similar results. Data in (<i>C</i>) and (<i>D</i>) are mean ± SEM from three independent experiments. *, <i>P</i> < 0.05 <i>vs</i>. siCL/control or untreated cells. #, <i>P</i> < 0.05 <i>vs</i>. siCL/leptin-treated cells.</p

STAT3-HDAC3 and HDAC4 are divergent pathways to mediate leptin-induced BMP-2 mRNA expression in human primary chondrocytes.

<p>Cells were kept as controls or treated with leptin for 1, 4, 8, and 24 h. (<i>A</i>) The associations of STAT3 with HDAC3 and HDAC4 were determined by an immunoprecipitation assay and Western blot and (<i>B</i>) the STAT3 acetylation was determined by Western blot. Results are representative of three independent experiments with similar results.</p

STAT3 regulates leptin-induced BMP-2 mRNA in human primary chondrocytes.

<p>(<i>A</i>) Cells were kept as controls or treated with leptin for 1, 4, 8, and 24 h, and the phosphorylations of STAT3 and STAT5 were determined by Western blot. (<i>B-C</i>) Cells were transfected with (<i>B</i>) ERK1- or ERK2-specific siRNA or (<i>C</i>) STAT3- or STAT5-specific siRNA and then kept as controls or treated with leptin for 4 h. (<i>B</i>) The phosphorylations of STAT3 and STAT5 were determined by Western blot and (<i>C</i>) the BMP-2 mRNA expression was determined by real-time PCR. (<i>D</i>) Cells were kept as controls or treated with leptin for 1, 4, and 8 h, and the STAT3-BMP-2 promoter binding activity was analyzed by chromatin immunoprecipitation and real-time PCR. Results in (<i>A</i>) and (<i>B</i>) are representative of three independent experiments with similar results. Data in (<i>C</i>) and (<i>D</i>) are mean ± SEM from three to four independent experiments. *, <i>P</i> < 0.05 <i>vs</i>. siCL/control or untreated cells. #, <i>P</i> < 0.05 <i>vs</i>. siCL/leptin-treated cells.</p

BMP-2 induction in leptin-stimulated human primary chondrocytes increases collagen II expression.

<p>(<i>A-B</i>) Cells were kept as controls or stimulated with leptin for 10’, 30’, 1, 4, 8, and 24 h. (<i>C-D</i>) Cells were (<i>C</i>) pretreated with Noggin or (<i>D</i>) transfected with BMP-2-, Smad1-, or Smad5-specific siRNA and then kept as controls or treated with leptin for 8 and 24 h. (<i>A</i>) The mRNA expressions of col1a1, runx2, and osteocalcin were determined by real-time PCR. (<i>B</i>-D) The protein expressions of collagen II and COX2 were determined by Western blot. Data in (<i>A</i>) are mean ± SEM from three independent experiments. Results in (<i>B-D</i>) are representative of three independent experiments with similar results. *, <i>P</i> < 0.05 <i>vs</i>. untreated cells. #, <i>P</i> < 0.05 <i>vs</i>. siCL/leptin-treated cells.</p

HDAC3/4 mediates leptin-induced BMP-2 mRNA expressions in human primary chondrocytes.

<p>(<i>A</i>) Cells were pretreated with DMSO or TSA and then kept as controls or treated with leptin for 4 and 8 h. The BMP-2 mRNA expression was determined by real-time PCR. (<i>B–C</i>). Cells were kept as controls or treated with leptin for 1, 4, 8 and 24 h. (<i>B</i>) The HDACs expressions were determined by Western blot. (<i>C</i>) The subcellular localization of HDAC3 and 4 were determined by cell fractionation assay and Western blot. (<i>D</i>) Cells were transfected with HDAC3- or HDAC4-specific siRNA and then kept as controls or treated with leptin for 4 h. The BMP-2 mRNA expression was determined by real-time PCR. Data in (<i>A</i>) and (<i>D</i>) are mean ± SEM from three independent experiments. *, <i>P</i> < 0.05 <i>vs</i>. DMSO/control, siCL/control or untreated cells. #, <i>P</i> < 0.05 <i>vs</i>. DMSO/ or siCL/leptin-treated cells. Results in (<i>B</i>) and (<i>C</i>) are representative of three independent experiments with similar results.</p

Schematic representation of the signaling pathways regulating leptin-induced BMP-2 and consequent collagen II expressions in human chondrocytes.

<p>Schematic representation of the signaling pathways regulating leptin-induced BMP-2 and consequent collagen II expressions in human chondrocytes.</p