Mechanical impact induces cartilage degradation via mitogen activated protein kinases

Abstract

Objective: To determine the activation of Mitogen activated protein (MAP) kinases in and around cartilage subjected to mechanical damage and to determine the effects of their inhibitors on impaction-induced chondrocyte death and cartilage degeneration.

Design: The phosphorylation of MAP kinases was examined with confocal microscopy and immunoblotting. The effects of MAP kinase inhibitors on impaction-induced chondrocyte death and proteoglycan (PG) loss were determined with fluorescent microscopy and 1, 9-Dimethyl-Methylene Blue (DMMB) assay. The expression of catabolic genes at mRNA levels was examined with quantitative real-time PCR.

Results: Early p38 activation was detected at 20 min and 1h post-impaction. At 24h, enhanced phosphorylation of p38 and extracellular signal-regulated protein kinase (ERK)1/2 was visualized in chondrocytes from in and around impact sites. The phosphorylation of p38 was increased by 3.0-fold in impact sites and 3.3-fold in adjacent cartilage. The phosphorylation of ERK-1 was increased by 5.8-fold in impact zone and 5.4-fold in adjacent cartilage; the phosphorylation of ERK-2 increased by 4.0-fold in impacted zone and 3.6-fold in adjacent cartilage. Furthermore, the blocking of p38 pathway did not inhibit impaction-induced ERK activation. The inhibition of p38 or ERK pathway significantly reduced injury-related chondrocyte death and PG losses. Quantitative Real-time PCR analysis revealed that blunt impaction significantly up-regulated matrix metalloproteinase (MMP)-13, Tumor necrosis factor (TNF)-α, and ADAMTS-5 expression.

Conclusion: These findings implicate p38 and ERK mitogen activated protein kinases (MAPKs) in the post-injury spread of cartilage degeneration and suggest that the risk of post-traumatic osteoarthritis (PTOA) following joint trauma could be decreased by blocking their activities, which might be involved in up-regulating expressions of MMP-13, ADAMTS-5, and TNF-α.

Figure 1

Osteochondral Explant Blunt Injury Model. (A) The image shows the gross appearance of a 14J/cm² blunt impact injury on the surface of a typical osteochondral explant. The center of the 2.5 cm × 2.5 cm explant was struck once with a 5-mm diameter platen. (B) Illustration of the method employed to harvest cartilage tissue from a traumatized explant. Six mm punches were used to harvest the impact site itself and 8 mm punches were used to harvest an adjacent annular ring of cartilage surrounding the impact site. (C) A high resolution scanned image of a safranin-O-, fast green-, and hematoxylin-stained section through the middle of an impact site. Blue arrows show the approximate boundaries of the platen contact. The surface is undamaged outside the contact area, but cartilage damage as superficial delamination and cracking are apparent within the impact site. The inset shows a close-up view of cracks running from the surface down to the transitional zone.

Figure 2

Effect of blunt impact on enhancing phosphorylation of p38 and ERK MAP kinases. Osteochondral explants were pre-equlibrated overnight in DMEM/F12/10% FBS prior to blunt impaction. Cartilage surfaces were subjected to a single blow with an energy of 14 J/cm². After 24 hrs, cartilage discs from the impacted zone and area immediately next to the zone were harvested with 5-mm biopsy punches. Right after harvesting, those discs were immersed into liquid nitrogen for at least 10 min before being transferred into −80 °C freezer for storage. Full thickness of cartilage discs were sectioned at 5 μm or 10 μm intervals. Cartilage sections were immediately fixed with 4% paraformaldehyde. Anti-phospho p38 or anti-phospho ERK1/2 antibody and Alexa Fluor 568 conjugated goat anti-rabbit antibody were used for indirectly staining of phosphorylated p38 or ERK MAP kinases in chondrocytes. Those sections were counter-stained with DAPI. A Zeiss 710 confocal microscope was utilized to image the distribution of phosphorylated p38 or ERK1/2 MAP kinases.

Figure 3

Short-term Effects of Blunt Impact on MAP kinases. Western blots show results for cartilage from non-impacted control explants (“C”) or from impact sites (“I”) and annulus sites (“A”) from injured explants. The samples were harvested at 20 min, 1 hr, and 3 hr post-impact or at equivalent culture times in controls. Blots were probed with antibodies recognizing total (t-) and phosphorylated (p-) forms of JNK1/2 and p38 (A) and ERK1/2 (B).

Figure 4

p38 and ERK MAP Kinase Activation at 12 and 24 hr Post-impact. Western blot results are shown for cartilage from non-impacted control explants (“C”), or from impact sites (“I”) and annulus sites (“A”) from injured explants. (A) Representative blots of extracts from cartilage harvested at 12 or 24 hours after impact. The blots were probed for total p38 or phosphorylated p38. (B) Fold increase in phosphorylation (impact or annulus versus control) based on densitometric analysis of western blots from at least 3 individual experiments. Error bars stand for 95% confidence intervals. (C) Western blots were probed for total and phosphorylated ERKs at 24 hrs in untreated control or impacted cartilage from five individual osteo-chondral explants. Results are shown for control, impact and annulus sits (“C”, “I”, “A”). (D) Average fold increase in phosphorylation (relative to non-impacted control) based on densitometric analysis from six individual experiments. Error bars show 95% confidence intervals.

Figure 5

Effect of blocking p38 MAP kinase activation on blunt impact-induced ERK activation. At 24 hr post-impaction, tissue lysates from non-impacted controls (N. C.), from impacted (I) or annulus (A) cartilage. Experimental groups included control explants without any inhibitor treatments, and explantspre-treated with U0126 (U.), a MEK1/2/ERK1/2 inhibitor, or with SB202190 (SB.), a p38 MAP kinase pathway inhibitor. Lysate proteins were resolved by SDS-PAGE and blotted onto a nitrocellulose membranes. The membranes were probed with anti-total ERK (t-ERK1/2) or anti-phospho-ERK (p-ERK1/2) antibodies. In these studies, two sets of osteo-chondral explants were pre-treated with above inhibitors. They are designated as U.-1, SB.-1, U.-2, and SB.-2 on the blots, respectively.

Figure 6

Effects of MAP Kinase Inhibitors on Chondrocyte Viability after Impact Injury. (A) Confocal microscope images show impact and annulus sites on explants stained with calcein AM and ethidium homodimer on day 7 post-impaction. Green-stained cells are viable and red stained cell are dead. The explants shown in the panels on the right were treated with 10 μM p38 MAP kinase inhibitor (+ SB202190). (B) The percentage of viable cells measured in non-impacted controls (N. C.), non-impacted controls treated with SB202190 only (SB90), impact sites in explants treated with SB202190 (SB90+I.,−I.) or annulus sites in treated explants (SB90+I.,−A.), and impact and annulus sites in untreated explants (Impact Only; I. O., −I.; I. O., −A.) (N=9; Error bars stand for 95% confidence intervals) (C) Percentage viability in non-impacted controls (N.C.), non-impacted controls treated with 10 μM ERK inhibitor, U0126, only (U0126) and impact and annulus sites treated with (U. + I., −I.; U. + I., −A.) or without UO126 (Impact Only; I. O., −I.; I. O., −A.) (N=9; Error bars stand for 95% confidence intervals). The viability of each group was compared to that of impact site from impacted explants without inhibitor treatment (Impact Only, I. O.).