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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-induced chemokine expression in chondrocytes causes FLS migration and invasion. a Migration and invasion of FLS treated (12 hours) with conditioned medium (CM) prepared from FLS infected with 800 MOI Ad-C or Ad-Epas1. Relative fold changes are presented (n = 6). b Migration and invasion of FLS treated (12 hours) with CM prepared from chondrocytes infected with 800 MOI Ad-C or Ad-Epas1. Relative fold changes are presented (n = 6). c Migration and invasion of FLS treated (12 hours) with CM prepared from WT (Epas1+/+) or Epas1+/− chondrocytes treated with IL-1β (2 ng/ml, 24 hours) (n = 4). d Migration of FLS treated with CM prepared from chondrocytes exposed to IL-1β (2 ng/ml, 24 hours) in the presence of IgG or neutralizing antibodies against CXCL2 and CCL5. Values presented as mean ± standard error of the mean (*P <0.05, **P <0.005). Scale bar: 50 μm. Ab antibody, CON control, FLS fibroblast-like synoviocytes, IL interleukin
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Fig5: HIF-2α-induced chemokine expression in chondrocytes causes FLS migration and invasion. a Migration and invasion of FLS treated (12 hours) with conditioned medium (CM) prepared from FLS infected with 800 MOI Ad-C or Ad-Epas1. Relative fold changes are presented (n = 6). b Migration and invasion of FLS treated (12 hours) with CM prepared from chondrocytes infected with 800 MOI Ad-C or Ad-Epas1. Relative fold changes are presented (n = 6). c Migration and invasion of FLS treated (12 hours) with CM prepared from WT (Epas1+/+) or Epas1+/− chondrocytes treated with IL-1β (2 ng/ml, 24 hours) (n = 4). d Migration of FLS treated with CM prepared from chondrocytes exposed to IL-1β (2 ng/ml, 24 hours) in the presence of IgG or neutralizing antibodies against CXCL2 and CCL5. Values presented as mean ± standard error of the mean (*P <0.05, **P <0.005). Scale bar: 50 μm. Ab antibody, CON control, FLS fibroblast-like synoviocytes, IL interleukin

Mentions: FLS, a major cell type in pannus tissue, migrate, attach to, invade, and destroy cartilage tissue by secreting proteases within the RA joints [4]. Since a major function of chemokines is recruitment of adjacent cells as a chemoattractant, we used an in vitro modified Boyden chamber assay to further examine whether increased expression of chemokines by HIF-2α in FLS and chondrocytes plays a role in FLS migration and invasion. Our data initially confirmed that CM from FLS infected with Ad-Epas1 stimulates both migration and invasion of FLS (Fig. 5a). Next, we examined whether HIF-2α-induced chemokines by chondrocytes also regulate migration and invasion of FLS. Notably, CM from Ad-Epas1-infected chondrocytes promoted both migration and invasion of FLS (Fig. 5b). The correlation between chondrocyte-originating HIF-2α and chemokine function in FLS migration and invasion was further validated. CM from chondrocytes treated with IL-1β stimulated the migration and invasion of WT (Epas1+/+) FLS (Fig. 5c). However, Epas1+/− FLS exhibited significantly reduced migration and invasion in the presence of IL-1β-treated chondrocyte CM (Fig. 5c). Furthermore, addition of anti-CXCL2 and anti-CCL5 antibodies to CM of IL-1β-treated chondrocytes blocked FLS migration (Fig. 5d). Based on the results, we propose that HIF-2α-induced chemokines by chondrocytes regulate FLS migration and invasion.Fig. 5


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-induced chemokine expression in chondrocytes causes FLS migration and invasion. a Migration and invasion of FLS treated (12 hours) with conditioned medium (CM) prepared from FLS infected with 800 MOI Ad-C or Ad-Epas1. Relative fold changes are presented (n = 6). b Migration and invasion of FLS treated (12 hours) with CM prepared from chondrocytes infected with 800 MOI Ad-C or Ad-Epas1. Relative fold changes are presented (n = 6). c Migration and invasion of FLS treated (12 hours) with CM prepared from WT (Epas1+/+) or Epas1+/− chondrocytes treated with IL-1β (2 ng/ml, 24 hours) (n = 4). d Migration of FLS treated with CM prepared from chondrocytes exposed to IL-1β (2 ng/ml, 24 hours) in the presence of IgG or neutralizing antibodies against CXCL2 and CCL5. Values presented as mean ± standard error of the mean (*P <0.05, **P <0.005). Scale bar: 50 μm. Ab antibody, CON control, FLS fibroblast-like synoviocytes, IL interleukin
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Fig5: HIF-2α-induced chemokine expression in chondrocytes causes FLS migration and invasion. a Migration and invasion of FLS treated (12 hours) with conditioned medium (CM) prepared from FLS infected with 800 MOI Ad-C or Ad-Epas1. Relative fold changes are presented (n = 6). b Migration and invasion of FLS treated (12 hours) with CM prepared from chondrocytes infected with 800 MOI Ad-C or Ad-Epas1. Relative fold changes are presented (n = 6). c Migration and invasion of FLS treated (12 hours) with CM prepared from WT (Epas1+/+) or Epas1+/− chondrocytes treated with IL-1β (2 ng/ml, 24 hours) (n = 4). d Migration of FLS treated with CM prepared from chondrocytes exposed to IL-1β (2 ng/ml, 24 hours) in the presence of IgG or neutralizing antibodies against CXCL2 and CCL5. Values presented as mean ± standard error of the mean (*P <0.05, **P <0.005). Scale bar: 50 μm. Ab antibody, CON control, FLS fibroblast-like synoviocytes, IL interleukin
Mentions: FLS, a major cell type in pannus tissue, migrate, attach to, invade, and destroy cartilage tissue by secreting proteases within the RA joints [4]. Since a major function of chemokines is recruitment of adjacent cells as a chemoattractant, we used an in vitro modified Boyden chamber assay to further examine whether increased expression of chemokines by HIF-2α in FLS and chondrocytes plays a role in FLS migration and invasion. Our data initially confirmed that CM from FLS infected with Ad-Epas1 stimulates both migration and invasion of FLS (Fig. 5a). Next, we examined whether HIF-2α-induced chemokines by chondrocytes also regulate migration and invasion of FLS. Notably, CM from Ad-Epas1-infected chondrocytes promoted both migration and invasion of FLS (Fig. 5b). The correlation between chondrocyte-originating HIF-2α and chemokine function in FLS migration and invasion was further validated. CM from chondrocytes treated with IL-1β stimulated the migration and invasion of WT (Epas1+/+) FLS (Fig. 5c). However, Epas1+/− FLS exhibited significantly reduced migration and invasion in the presence of IL-1β-treated chondrocyte CM (Fig. 5c). Furthermore, addition of anti-CXCL2 and anti-CCL5 antibodies to CM of IL-1β-treated chondrocytes blocked FLS migration (Fig. 5d). Based on the results, we propose that HIF-2α-induced chemokines by chondrocytes regulate FLS migration and invasion.Fig. 5

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