• IMMUNE NETWORK
• /
• v.6 no.4
• /
• pp.204-210
• /
• 2006

Background: Nitric oxide (NO) in articular chondrocytes regulates dedifferentiation and inflammatory responses by modulating MAP kinases. In this study, we investigated whether the Src kinase in chondrocytes regulates NO-induced dedifferentiation and cyclooxygenase-2 (COX-2) expression. Methods: Primary chondrocytes were treated with various concentrations of SNP for 24 h. The COX-2 and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin $E_2\;(PGE_2)$ was determined by using a $PGE_2$ assay kit. Expression and distribution of p-Caveolin and COX-2 in rabbit articular chondrocytes and cartilage explants were determined by immunohistochemical staining and immunocytochemical staining, respectively. Results: SNP treatment stimulated Src kinase activation in a dose-dependent manner in articular chondrocytes. The Src kinase inhibitors PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine], a significantly blocked SNP-induced p38 kinase and caveolin-1 activation in a dose-dependent manner. Therefore, to determine whether Src kinase activation is associated with dedifferentiation and/or COX-2 expression and $PGE_2$ production. As expected, PP2 potentiated SNP-stimulated dedifferentiation, but completely blocked both COX-2 expression and $PGE_2$ production. And also, levels of p-Caveolin and COX-2 protein expression were increased in SNP-treated primary chondrocytes and osteoarthritic and rheumatoid arthritic cartilage, suggesting that p-Caveolin may playa role in the inflammatory responses of arthritic cartilage. Conclusion: Our previously studies indicated that NO caused dedifferentiation and COX-2 expression is regulated by p38 kinase through caveolin-1 (1). Therefore, our results collectively suggest that Src kinase regulates NO-induced dedifferentiation and COX-2 expression in chondrocytes via p38 kinase in association with caveolin-1.

• The Journal of Korean Medicine
• /
• v.27 no.1 s.65
• /
• pp.220-228
• /
• 2006

Objectives : Interleukin-1 (IL-1)${\beta}$ in articular chondrocytes regulates differentiation, apoptosis, and inflammatory responses. It is still controversial, So, we investigated IL- $1{\beta}$ induces chondrocytes dedifferentiation and death. Also, we studied the role of the mitogen-activated protein kinase (MAPK) subtypes on IL-$1{\beta}$-induced dedifferentiation and apoptosis. Methods : To evaluation of dedifferentiation by chemokines of chondrocytes, we assessed such as proteoglycan, collagen, MMP-3 and MMP-13 by RT-PCR analysis. Also, to assess of apoptosis effect by chemokines, we measured annexin V/propidium iodode (PI) and sub G1 cells in chondrocytes by flowcytometric analysis Results : IL-$1{\beta}$ treatment did not affect activation of ERK-1/2, but stimulation of p38 kinase. Inhibition of phospho ERK-1/2 with PD98059 enhanced IL-1b-induced dedifferentiation, and apoptosis up to 13.5%, whereas inhibition of phospho p38 kinase with SB203580 inhibited dedifferentiation, and apoptosis. Conclusions : Our results indicate that SB203580, p38 kinase inhibitor, inhibits IL-$1{\beta}$-induced dedifferentiation, and apoptosis by the inhibition of type II collagen expression and proteoglycan synthesis of rabbit articular chondrocytes.

• Biomedical Science Letters
• /
• v.15 no.4
• /
• pp.343-347
• /
• 2009

Cytochalasin D (CD) is known as a disruptor of actin cytoskeleton architecture in chondrocytes. We have studied the role of CD in retinoic acid (RA) caused dedifferentiation and inflammation responses in rabbit articular chondrocytes. We have examined the effect of CD on RA induced dedifferentiation of chondrocytes. CD inhibited RA induced dedifferentiation determined by Western blot analysis and Alcian blue staining in rabbit articular chondrocytes. Also, CD additionally reduced inflammation response molecules such as cyclooxygenase-2 (COX-2) and prostaglandin $E_2$ ($PGE_2$) in RA treated cells. Treatment of CD reduced phosphorylation of p38 by treatment of RA. Inhibiton of p38kinase with SB203580 reduced expression of COX-2 and production of $PGE_2$ by treatment of CD in RA treated cells. But, Inhibiton of p38kinase with SB203580 did not any relationship with effect of CD on RA caused dedifferentiation. In summary, our results indicate that CD regulates RA reduced expression of COX-2 and production of PGE2 via p38kinase pathway.

• Animal cells and systems
• /
• v.14 no.2
• /
• pp.73-81
• /
• 2010

In this study, monoclonal antibodies against lysosomal acid phosphatase (LAP) of a salamander, Hynobius leechii, were used to determine the spatial and temporal expression of the LAP in the regenerating limbs. The Western blot and immunohistochemical analysis in the limb regeneration revealed that LAP was highly expressed at the dedifferentiation stage, especially in the wound epidermis and dedifferentiating limb tissues such as muscle and cartilage. With RA treatment, the LAP expression became upregulated in terms of both level and duration in the wound epidermis, blastemal cell and dedifferentiating limb tissues. In addition, in situ activity staining of LAP showed a similar result to that of immunohistochemistry. Thus, the activity profile of LAP activity coincides well with the expression profile of LAP during the dedifferentiation period. Furthermore, to examine the effects of lysosomal enzymes including LAP on salamander limb regeneration, lysosome extract was microinjected into limb regenerates. Interestingly, when the lysosome extract was microinjected into limb regenerates with a low dose of RA($50\;{\mu}g/g$ body wt.), skeletal pattern duplication occurred frequently in the proximodistal and transverse axes. Therefore, lysosomal enzymes might cause the regenerative environment and RA plays dual roles in the modification of positional value as well as evocation of extensive dedifferentiation for pattern duplication. In conclusion, these results support the hypothesis that dedifferentiation is a crucial event in the process of limb regeneration and RA-evoked pattern duplication, and lysosomal enzymes may play important role(s) in this process.

• Journal of Life Science
• /
• v.21 no.4
• /
• pp.534-541
• /
• 2011

Retinoic acid (RA), a metabolite of vitamin A, is known to regulate dedifferentiation of rabbit articular chondrocytes. The regulatory mechanism of dedifferentiation by RA is not yet understood. Thus, the effect of RA on the regulation of nitric oxide (NO)-induced dedifferentiation was investigated in rabbit articular chondrocytes. RA caused loss of the differentiated chondrocyte phenotype as demonstrated by inhibition of type II collagen expression and proteoglycan synthesis. RA also accelerated NO-induced dedifferentiation in rabbit articular chondrocytes as detected by expression of type II collagen and Sox-9 using Western blot analysis and production of sulfated proteoglycan using Alcain blue staining. Further, RA potentiated NO-induced activation of ERK. Inhibition of ERK with PD98059 (PD) recovered the expression of type II collagen and Sox-9 and production of sulfate proteoglycan in NO-induced dedifferentiated chondrocytes by RA treatment. Our findings suggest that RA accelerates NO-induced dedifferentiation of rabbit articular chondrocytes via the ERK pathway.

• Animal cells and systems
• /
• v.4 no.2
• /
• pp.145-150
• /
• 2000

Retinoic acid (RA) evokes pattern duplication in the regenerating salamander limb. Interestingly, it also enhances dedifferentiation in the regenerate by the morphological, histological and biochemical criteria. To examine whether there is any correlation between the RA-evoked pattern duplication and de novo protein synthetic profile in the regenerating salamander limb, especially during dedifferentiation, we analyzed stage-specific protein synthesis pattern in the normal and RA-treated regenerating limbs by metabolic labeling followed by two-dimensional gel electrophoresis. In the regenerating limbs without RA treatment, a few hundred kinds of proteins were found to be synthesized at the stage of wound healing and the total number of protein synthesized increased greatly as regeneration proceeded. The same trend was also observed in the RA-treated regenerating limbs. Interestingly, some protein spots were noted to be either newly synthesized or highly expressed by the RA treatment especially at the stage of dedifferentiation. The results shows that the enhancement of dedifferentiation state after the RA treatment correlates well with the protein synthesis profile, and suggest that those proteins are important for the RA-evoked pattern duplication in the regenerating limbs of salamander.

• IMMUNE NETWORK
• /
• v.6 no.3
• /
• pp.117-122
• /
• 2006

Background: Caveolin, a family of integral membrane proteins are a principal component of caveolae membranes. In this study, we investigated the effect of p38 kinase on differentiation and on inflammatory responses in sodium nitroprusside (SNP)-treated chondrocytes. Methods: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. SNP was used as a nitric oxide (NO) donor. In this experiments measuring SNP dose response, primary chondrocytes were treated with various concentrations of SNP for 24h. The time course of the SNP response was determined by incubating cells with 1mM SNP for the indicated time period $(0{\sim}24h)$. The cyclooxygenase-2 (COX-2) and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin $E_2\;(PGE_2)$ assay was used to measure the COX-2 activity. The tyrosine phosphorylation of caveolin-1 was determined by immunoblot analysis and immunostaining. Results: SNP treatment stimulated tyrosine phosphorylation of caveolin-1 and activation of p38 kinase. SNP additionally caused dedifferentiation and inflammatory response. We showed previously that SNP treatment stimulated activation of p38 kinase and ERK-1/-2. Inhibition of p38 kinase with SB203580 reduced caveolin-1 tyrosine phosphorylation and COX-2 expression but enhanced dedifferentiation, whereas inhibition of ERK with PD98059 did not affect caveolin-1 tyrosine phosphorylation levels, suggesting that ERK at least is not related to dedifferentiation and COX-2 expression through caveolin-1 tyrosine phosphorylation. Conclusion: Our results indicate that SNP in articular chondrocytes stimulates dedifferentiation and inflammatory response via p38 kinase signaling in association with caveolin-1 phosphorylation.

• The Korean Journal of Zoology
• /
• v.39 no.1
• /
• pp.65-74
• /
• 1996

Treatment of regenerating amphibian limbs with retinoic acid (RA) is known to induce paftern duplication, which is closely related to the extent of dedifferentiation. In the present study, the activities of trypsin- and chymotrypsin-like proteases are examined to delineate a possible role in the process of dedifferentiation in the regenerating limbs of urodeles, the Korean salamander (Hynobius leechii) and the Mexican axolod (Ambystoma mexicanum). Specifically, we were interested to know if there is any correlation between trypsin- and chymotrypsin-like protease activities and the state of dedifferentiation which is augmented by RA treatment. We were also interested in expoloring if there is any species-specific difference in the profile of enzyme activities during limb regeneration. The results showed that the activities of these two enzymes reached a peak level at dedifferentiation stage, and RA treatment caused elevation of their activities, especially in the case of trypsin-like protease. The increase of trypsin-like protease activity after RA treatment was pronounced in the Korean salamander, which might reflect a species-specific responsiveness to RA. The present results imply that trypsin and chymotrypsin or similar proteases may play an active role in the process of dedifferentiation in regenerating limbs, and that trypsin or trypsin-like eryrymes might be involved in the RA-evoked enhancement of dedifferentiation which precedes overt pattern duplication.

• Animal cells and systems
• /
• v.16 no.6
• /
• pp.462-468
• /
• 2012

Urodeles including newt and salamander have remarkable regenerative capacity during the postembryonic life span. Some of the unique features are the formation of the well-developed wound epidermis and the active dedifferentiation process in the early phase of regeneration. These are regarded as key events for the successful regeneration since no further regenerative activity is possible without them. In this study, we investigated the role of retinoic acid (RA) in salamander limb regeneration by blocking RA synthesis using disulfiram, an inhibitor of aldehyde dehydrogenase that oxidizes retinal to RA. Disulfiram treatment resulted in delaying the limb regeneration processes via inhibition of wound epidermis formation and dedifferentiation process. When RA was administered after disulfiram treatment, the inhibitory effect of disulfiram was rescued. In addition, disulfiram treatment after the dedifferentiation stage resulted in the mild retardation of limb regeneration, suggesting that RA might also be involved in the blastema outgrowth. Furthermore, salamander MMP-9 gene expression was also inhibited by disulfiram treatment. Collectively, our findings indicate that endogenous RA may play an important role(s) in the early phase of limb regeneration by regulating the expression of molecules responsible for the modification of intracellular and extracellular environment during salamander limb regeneration.

• Development and Reproduction
• /
• v.2 no.1
• /
• pp.53-62
• /
• 1998

The lysosomal acid hydrolases including lysosomal acid phosphatase (LAP) are believed to play an important role in intracellular and extracellular degradation. LAP was reported to increase its activity in dedifferentiation stage during urodele limb regeneration. In the paresent study, LAP localization in the Mexican axolotl (Ambystoma mexicanum) limb regenerates was investigated by immunohistochemistry. LAP immunoreactivity with monoclonal antibody against Korean salamander (Hynobius leehii) LAP was observed mainly in the wound epidermis, blastema cells, muscle, and cartilage which were under dedifferentiation process in axolotl limb regenerates. Moreover, LAP immunoreactivity increased gradually during the early phase of lib regeneration and reached the peak level at dedifferentiation stage. However, as redifferentiation begans, LAP immunoreactivity decreased slowly to the basal level. Retinoic acid (RA) which is known to induce skeleton pattern duplication in regenerating urodele limb appears to enhance LAP immunoreactivity. In the RA-treate limg regenerates, LAP immunoreactivity was higher than in the normal regenerates. In addition, the LAP expression period was more extended in the RA treated regenerates than in the normal regenerates. These results suggest that RA is involved in the extension of dedifferentiation state in RA-treated limb regenerate.