Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells

Abstract

Background: Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/β-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells.

Methods: Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and β-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE₂) levels were measured using enzyme-linked immunosorbent assay.

Results: Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and β-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE₂, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered.

Conclusions: PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on β-catenin in the canonical Wnt pathway was limited. β-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE₂ owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

Keywords: Disc degeneration; Matrix metalloproteinase; Nucleus pulposus; Phorbol myristate acetate; Protein kinase C-delta.

Fig. 1. Western blot analysis of the expression of protein kinase C (PKC)-δ (A) and β-catenin and its phosphorylated form (B). Values are presented as mean ± standard deviation. PMA: phorbol myristate acetate, BII: bisindolylmaleimide I. A p-value < 0.05 was considered significant.

Fig. 2. Glycosaminoglycan (GAG) content in control and phorbol myristate acetate (PMA)-treated cells. (A) Total sulfated GAG. (B) Chondroitin-4 sulfate expression. Values are presented as mean ± standard deviation. BII: bisindolylmaleimide I. A p-value < 0.05 was considered significant.

Fig. 3. Effect of phorbol myristate acetate (PMA) on the expression of collagen type II. (A) Expression of collagen type II messenger ribonucleic acid. (B) Expression of collagen type II protein. Values are presented as mean ± standard deviation. BII: bisindolylmaleimide I. A p-value < 0.05 was considered significant.

Fig. 4. Effect of phorbol myristate acetate (PMA) on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs). (A) Western blot analysis of the expression of MMP1, MMP13, and TIMP1. (B) Western blot and immunofluorescent images showing the expression of a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) (the red, green, and blue colors indicate actin, ADAMTS5, and the nucleus, respectively). Values are presented as mean ± standard deviation. BII: bisindolylmaleimide I. A p-value < 0.05 was considered significant.

Fig. 5. Expression of interleukin-6 (IL-6) and prostaglandin E2 (PGE₂) in control and phorbol myristate acetate (PMA)-treated nucleus pulposus cells. (A) Analysis of IL-6 expression using western blotting. (B) Analysis of IL-6 and PGE₂ expression using enzyme-linked immunosorbent assay. Values are presented as mean ± standard deviation. BII: bisindolylmaleimide I. A p-value < 0.05 was considered significant.

Fig. 6. Expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphorylated NF-κB (pNF-κB) in the control and phorbol myristate acetate (PMA)-treated nucleus pulposus (NP) cells. (A) Western blot analysis of the expression of NF-κB and its phosphorylated form (pNF-κB). (B) Immunofluorescent images showing NF-κB expression in NP cells (magnification: 20× or 40×; the red and blue colors indicate NF-κB and the nucleus, respectively). Values are presented as mean ± standard deviation. BII: bisindolylmaleimide I, C: cytosolic, N: nuclear. A p-value < 0.05 was considered significant.

Fig. 7. Effect of phorbol myristate acetate (PMA) treatment on transient receptor potential vanilloid 4 (TRPV4) expression in nucleus pulposus (NP) cells. (A) Analysis of TRPV4 expression using Western blot. (B) Immunofluorescent images showing TRPV4 expression in NP cells (magnification: 20× or 40×; the red and blue colors indicate TRPV4 and the nucleus, respectively). Values are presented as mean ± standard deviation. BII: bisindolylmaleimide I. A p-value < 0.05 was considered significant.