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HIF-2α-induced chemokines stimulate motility of fibroblast-like synoviocytes and chondrocytes into the cartilage-pannus interface in experimental rheumatoid arthritis mouse models.

Huh YH, Lee G, Lee KB, Koh JT, Chun JS, Ryu JH - Arthritis Res. Ther. (2015)

Bottom Line: Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR.HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue.HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS.

View Article: PubMed Central - PubMed

Affiliation: BioImaging and Cell Dynamics Research Center, Gwangju Institute of Science and Technology, 123 Cheomdangwagi-ro, Buk-gu, Gwangju, 61005, Republic of Korea. yhuh@gist.ac.k.

ABSTRACT

Introduction: Pannus formation and resulting cartilage destruction during rheumatoid arthritis (RA) depends on the migration of synoviocytes to cartilage tissue. Here, we focused on the role of hypoxia-inducible factor (HIF)-2α-induced chemokines by chondrocytes in the regulation of fibroblast-like synoviocyte (FLS) migration into the cartilage-pannus interface and cartilage erosion.

Methods: Collagen-induced arthritis (CIA), K/BxN serum transfer, and tumor necrosis factor-α transgenic mice were used as experimental RA models. Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR. FLS motility was evaluated using transwell migration and invasion assays. The specific role of HIF-2α was determined via local deletion of HIF-2α in joint tissues or using conditional knockout (KO) mice. Cartilage destruction, synovitis and pannus formation were assessed via histological analysis.

Results: HIF-2α and various chemokines were markedly upregulated in degenerating cartilage and pannus of RA joints. HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue. HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS. Local deletion of HIF-2α in joint tissues inhibited pannus formation adjacent to cartilage tissue and cartilage destruction caused by K/BxN serum transfer. Furthermore, conditional knockout of HIF-2α in cartilage blocked pannus formation in adjacent cartilage but not bone tissue, along with inhibition of cartilage erosion caused by K/BxN serum transfer.

Conclusion: Our findings suggest that chemokines induced by IL-1β or HIF-2α in chondrocytes regulate pannus expansion by stimulating FLS migration and invasion, leading to cartilage erosion during RA pathogenesis.

No MeSH data available.


Related in: MedlinePlus

HIF-2α mediates IL-1β-induced chemokine expression in chondrocytes. a RT-PCR and b qRT-PCR analyses of mRNA in chondrocytes treated with IL-1β for 24 hours (n = 5). c RT-PCR and western blot analyses of the indicated molecules in primary cultures of chondrocytes isolated from WT (Epas1+/+) or Epas1+/− mice treated with IL-1β for 24 hours. RT-PCR was carried out to detect transcript levels of Epas1, Cxcl1, Cxcl2, and Ccl5. Secreted chemokine proteins were examined via western blot. d Immunostaining of HIF-2α, CXCL1, CXCL2, and CCL5 in cartilage tissue of WT and Epas1+/− DBA1/J mice with CIA. Values presented as mean ± standard error of the mean (*P <0.05, **P <0.01). Scale bar: 50 μm. CIA collagen-induced arthritis, DAPI 4′,6-diamidino-2-phenylindole, HIF hypoxia-inducible factor, IL interleukin
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Fig4: HIF-2α mediates IL-1β-induced chemokine expression in chondrocytes. a RT-PCR and b qRT-PCR analyses of mRNA in chondrocytes treated with IL-1β for 24 hours (n = 5). c RT-PCR and western blot analyses of the indicated molecules in primary cultures of chondrocytes isolated from WT (Epas1+/+) or Epas1+/− mice treated with IL-1β for 24 hours. RT-PCR was carried out to detect transcript levels of Epas1, Cxcl1, Cxcl2, and Ccl5. Secreted chemokine proteins were examined via western blot. d Immunostaining of HIF-2α, CXCL1, CXCL2, and CCL5 in cartilage tissue of WT and Epas1+/− DBA1/J mice with CIA. Values presented as mean ± standard error of the mean (*P <0.05, **P <0.01). Scale bar: 50 μm. CIA collagen-induced arthritis, DAPI 4′,6-diamidino-2-phenylindole, HIF hypoxia-inducible factor, IL interleukin

Mentions: IL-1β is one of the major proinflammatory cytokines regulating RA pathogenesis [26]. IL-1β also transcriptionally upregulates HIF-2α in FLS and chondrocytes [7, 21]. Accordingly, we examined whether IL-1β stimulates chemokine expression via the HIF-2α pathway in chondrocytes. IL-1β enhanced chemokine expression in WT chondrocytes (Fig. 4a, b). This upregulation was markedly inhibited in Epas1+/− chondrocytes (Fig. 4c). We previously showed that deletion of one allele of Epas1 significantly inhibits the hallmarks of RA, including synovitis, cartilage erosion, and pannus formation [7]. Consistently, Epas1+/− DBA/1 J mice under CIA conditions showed marked reduction of CXCL1, CXCL2, and CCL5 expression, compared with their corresponding WT littermates (Fig. 4d). These findings suggest that regulation of chemokines in RA cartilage is controlled by HIF-2α.Fig. 4


HIF-2α-induced chemokines stimulate motility of fibroblast-like synoviocytes and chondrocytes into the cartilage-pannus interface in experimental rheumatoid arthritis mouse models.

Huh YH, Lee G, Lee KB, Koh JT, Chun JS, Ryu JH - Arthritis Res. Ther. (2015)

HIF-2α mediates IL-1β-induced chemokine expression in chondrocytes. a RT-PCR and b qRT-PCR analyses of mRNA in chondrocytes treated with IL-1β for 24 hours (n = 5). c RT-PCR and western blot analyses of the indicated molecules in primary cultures of chondrocytes isolated from WT (Epas1+/+) or Epas1+/− mice treated with IL-1β for 24 hours. RT-PCR was carried out to detect transcript levels of Epas1, Cxcl1, Cxcl2, and Ccl5. Secreted chemokine proteins were examined via western blot. d Immunostaining of HIF-2α, CXCL1, CXCL2, and CCL5 in cartilage tissue of WT and Epas1+/− DBA1/J mice with CIA. Values presented as mean ± standard error of the mean (*P <0.05, **P <0.01). Scale bar: 50 μm. CIA collagen-induced arthritis, DAPI 4′,6-diamidino-2-phenylindole, HIF hypoxia-inducible factor, IL interleukin
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Fig4: HIF-2α mediates IL-1β-induced chemokine expression in chondrocytes. a RT-PCR and b qRT-PCR analyses of mRNA in chondrocytes treated with IL-1β for 24 hours (n = 5). c RT-PCR and western blot analyses of the indicated molecules in primary cultures of chondrocytes isolated from WT (Epas1+/+) or Epas1+/− mice treated with IL-1β for 24 hours. RT-PCR was carried out to detect transcript levels of Epas1, Cxcl1, Cxcl2, and Ccl5. Secreted chemokine proteins were examined via western blot. d Immunostaining of HIF-2α, CXCL1, CXCL2, and CCL5 in cartilage tissue of WT and Epas1+/− DBA1/J mice with CIA. Values presented as mean ± standard error of the mean (*P <0.05, **P <0.01). Scale bar: 50 μm. CIA collagen-induced arthritis, DAPI 4′,6-diamidino-2-phenylindole, HIF hypoxia-inducible factor, IL interleukin
Mentions: IL-1β is one of the major proinflammatory cytokines regulating RA pathogenesis [26]. IL-1β also transcriptionally upregulates HIF-2α in FLS and chondrocytes [7, 21]. Accordingly, we examined whether IL-1β stimulates chemokine expression via the HIF-2α pathway in chondrocytes. IL-1β enhanced chemokine expression in WT chondrocytes (Fig. 4a, b). This upregulation was markedly inhibited in Epas1+/− chondrocytes (Fig. 4c). We previously showed that deletion of one allele of Epas1 significantly inhibits the hallmarks of RA, including synovitis, cartilage erosion, and pannus formation [7]. Consistently, Epas1+/− DBA/1 J mice under CIA conditions showed marked reduction of CXCL1, CXCL2, and CCL5 expression, compared with their corresponding WT littermates (Fig. 4d). These findings suggest that regulation of chemokines in RA cartilage is controlled by HIF-2α.Fig. 4

Bottom Line: Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR.HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue.HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS.

View Article: PubMed Central - PubMed

Affiliation: BioImaging and Cell Dynamics Research Center, Gwangju Institute of Science and Technology, 123 Cheomdangwagi-ro, Buk-gu, Gwangju, 61005, Republic of Korea. yhuh@gist.ac.k.

ABSTRACT

Introduction: Pannus formation and resulting cartilage destruction during rheumatoid arthritis (RA) depends on the migration of synoviocytes to cartilage tissue. Here, we focused on the role of hypoxia-inducible factor (HIF)-2α-induced chemokines by chondrocytes in the regulation of fibroblast-like synoviocyte (FLS) migration into the cartilage-pannus interface and cartilage erosion.

Methods: Collagen-induced arthritis (CIA), K/BxN serum transfer, and tumor necrosis factor-α transgenic mice were used as experimental RA models. Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR. FLS motility was evaluated using transwell migration and invasion assays. The specific role of HIF-2α was determined via local deletion of HIF-2α in joint tissues or using conditional knockout (KO) mice. Cartilage destruction, synovitis and pannus formation were assessed via histological analysis.

Results: HIF-2α and various chemokines were markedly upregulated in degenerating cartilage and pannus of RA joints. HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue. HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS. Local deletion of HIF-2α in joint tissues inhibited pannus formation adjacent to cartilage tissue and cartilage destruction caused by K/BxN serum transfer. Furthermore, conditional knockout of HIF-2α in cartilage blocked pannus formation in adjacent cartilage but not bone tissue, along with inhibition of cartilage erosion caused by K/BxN serum transfer.

Conclusion: Our findings suggest that chemokines induced by IL-1β or HIF-2α in chondrocytes regulate pannus expansion by stimulating FLS migration and invasion, leading to cartilage erosion during RA pathogenesis.

No MeSH data available.


Related in: MedlinePlus