Pomegranate extract inhibits the interleukin-1β-induced activation of MKK-3, p38α-MAPK and transcription factor RUNX-2 in human osteoarthritis chondrocytes.
BACKGROUND
Pomegranate has been revered throughout history for its medicinal properties. p38-MAPK is a major signal-transducing pathway in osteoarthritis (OA) and its activation by interleukin-1β (IL-1β) plays a critical role in the expression and production of several mediators of cartilage catabolism in OA. In this study we determined the effect of a standardized pomegranate extract (PE) on the IL-1β-induced activation of MKK3/6, p38-MAPK isoforms and the activation of transcription factor RUNX-2 in primary human OA chondrocytes.


METHODS
Human chondrocytes were derived from OA cartilage by enzymatic digestion, treated with PE and then stimulated with IL-1β. Gene expression of p38-MAPK isoforms was measured by RT-PCR. Western immunoblotting was used to analyze the activation of MAPKs. Immunoprecipitation was used to determine the activation of p38-MAPK isoforms. DNA binding activity of RUNX-2 was determined using a highly sensitive and specific ELISA. Pharmacological studies to elucidate the involved pathways were executed using transfection with siRNAs.


RESULTS
Human OA chondrocytes expressed p38-MAPK isoforms p38α, -γ and -δ, but not p38β. IL-1β enhances the phosphorylation of the p38α-MAPK and p38γ-MAPK isoforms but not of p38δ-MAPK isoform in human OA chondrocytes. Activation of p38-MAPK in human OA chondrocytes was preferentially mediated via activation of MKK3. In addition, we also demonstrate that polyphenol rich PE inhibited the IL-1β-induced activation of MKK3, p38α-MAPK isoform and DNA binding activity of the transcription factor RUNX-2.


CONCLUSIONS
Our results provide an important insight into the molecular basis of the reported cartilage protective and arthritis inhibitory effects of pomegranate extract. These novel pharmacological actions of PE on IL-1β stimulated human OA chondrocytes impart a new suggestion that PE or PE-derived compounds may be developed as MKK and p38-MAPK inhibitors for the treatment of OA and other degenerative/inflammatory diseases.
Department of Medicine, Division of Rheumatology, MetroHealth Medical Center/Case Western Reserve University, 2500 Metro Health Drive, Cleveland, OH 44109, USA. zrasheed@metrohealth.org
Zafar
Z
Rasheed
Nahid
N
Akhtar
Tariq M
TM
Haqqi
D000368:Aged; D015153:Blotting, Western; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects*; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D047468:Immunoprecipitation; D053583:Interleukin-1beta / Q000378:metabolism; D029561:Lythraceae / Q000737:chemistry / Q000378:metabolism*; D048371:MAP Kinase Kinase 3 / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000378:metabolism*; D010936:Plant Extracts / Q000494:pharmacology*; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D050659:Core Binding Factor Alpha 1 Subunit; D053583:Interleukin-1beta; D010936:Plant Extracts; D034741:RNA, Small Interfering; C493709:RUNX2 protein, human; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3