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Dickkopf‑related protein 1 induces angiogenesis by upregulating vascular endothelial growth factor in the synovial fibroblasts of patients with temporomandibular joint disorders.

Jiang SJ, Li W, Li YJ, Fang W, Long X - Mol Med Rep (2015)

Bottom Line: The present study also investigated the effects of DKK‑1 on the migration of human umbilical vein endothelial cells (HUVEC).In addition, DKK‑1 induced HUVEC migration, and HIF‑1α siRNA inhibited DKK‑1‑induced cell migration.Furthermore, HIF‑1α may be associated with DKK‑1‑induced HUVEC activation.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‑MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079, P.R. China.

ABSTRACT
Angiogenesis has an important role in the progression of temporomandibular joint disorders (TMD). The aim of the present study was to explore the association between dickkopf‑related protein 1 (DKK‑1) and angiogenesis in TMD. The expression levels of DKK‑1 and vascular endothelial growth factor (VEGF) were quantified by an ELISA assay of the synovial fluid from patients with TMD. The correlation between DKK‑1 and VEGF was analyzed by Pearson correlation test. Synovial fibroblasts were isolated from patients with TMD and were subsequently treated with recombinant human DKK‑1, anti‑DKK‑1 antibody, hypoxia inducible factor‑1α (HIF‑1α), or small interfering RNA (siRNA). The expression levels of DKK‑1, HIF‑1α, and VEGF were subsequently quantified. The present study also investigated the effects of DKK‑1 on the migration of human umbilical vein endothelial cells (HUVEC). Increased expression levels of DKK‑1 were concordant with increased expression levels of VEGF in the synovial fluid from patients with TMD. In the synovial fibroblasts, DKK‑1 increased the expression levels of VEGF, and promoted HIF‑1α nuclear localization. In addition, DKK‑1 induced HUVEC migration, and HIF‑1α siRNA inhibited DKK‑1‑induced cell migration. The results of the present study indicate that DKK‑1 is associated with angiogenesis in the synovial fluid of patients with TMD. Furthermore, HIF‑1α may be associated with DKK‑1‑induced HUVEC activation.

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Hypoxia inducible factor-1α (HIF-1α) mediates dickkopf-related protein 1 (DKK-1) regulation of vascular endothelial growth factor (VEGF) expression. (A) Western blotting demonstrated that the protein expression levels of HIF-1α in the synovial fibroblasts transiently transfected with scramble or HIF-1α small interfering (si)RNA 1 and 2. The proteins were extracted 72 h post-transfection. (B) HIF-1α siRNA (HIF-1α siRNA 2 as a target sequence) significantly inhibited the upregulatory effects of recombinant human (rh)DKK-1 on VEGF protein expression in synovial fibroblasts, as demonstrated by western blot analysis. (C and D) HIF-1α siRNA also inhibited the upregulatory effects of rhDKK-1 on the mRNA expression levels of VEGF in synovial fibroblasts. The mRNA expression levels of HIF-1α exhbited no significant changes between the control and rhDKK-1 treatment group. The results are presented as the mean ± standard error of the mean. **P<0.01, and ***P<0.001; one-way analysis of variance. RNA was extracted 48 h post-transfection. (E) Synovial fibroblasts treated with 10 ng/ml rhDKK-1 for 24 h exhibited HIF-1α nuclear translocation, as determined by immunofluorescence. Scale bars, 25 µm. (F) Quantification of nuclear translocation of HIF-1α in the vehicle group and rhDKK-1-treatment group. The results are presented as the mean ± standar error of the mean, **P<0.01; Student's t-test.
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f3-mmr-12-04-4959: Hypoxia inducible factor-1α (HIF-1α) mediates dickkopf-related protein 1 (DKK-1) regulation of vascular endothelial growth factor (VEGF) expression. (A) Western blotting demonstrated that the protein expression levels of HIF-1α in the synovial fibroblasts transiently transfected with scramble or HIF-1α small interfering (si)RNA 1 and 2. The proteins were extracted 72 h post-transfection. (B) HIF-1α siRNA (HIF-1α siRNA 2 as a target sequence) significantly inhibited the upregulatory effects of recombinant human (rh)DKK-1 on VEGF protein expression in synovial fibroblasts, as demonstrated by western blot analysis. (C and D) HIF-1α siRNA also inhibited the upregulatory effects of rhDKK-1 on the mRNA expression levels of VEGF in synovial fibroblasts. The mRNA expression levels of HIF-1α exhbited no significant changes between the control and rhDKK-1 treatment group. The results are presented as the mean ± standard error of the mean. **P<0.01, and ***P<0.001; one-way analysis of variance. RNA was extracted 48 h post-transfection. (E) Synovial fibroblasts treated with 10 ng/ml rhDKK-1 for 24 h exhibited HIF-1α nuclear translocation, as determined by immunofluorescence. Scale bars, 25 µm. (F) Quantification of nuclear translocation of HIF-1α in the vehicle group and rhDKK-1-treatment group. The results are presented as the mean ± standar error of the mean, **P<0.01; Student's t-test.

Mentions: To investigate the mechanisms underlying DKK-1 regulation of VEGF expression, the expression levels of HIF-1α, an upstream inducer of VEGF, were analyzed. HIF-1α siRNA 2 markedly inhibited the expression of HIF-1α (Fig. 3A). HIF-1α siRNA 2 was therefore chosen as the target sequence. As shown in Fig. 3B, the protein expression levels of HIF-1α were increased in synovial fibroblasts treated with rhDKK-1, as compared with the negative control. The protein expression levels of VEGF exhibited similar results. However, the upregulatory effects of rhDKK-1 on VEGF and HIF-1α protein expression were inhibited by HIF-1α siRNA (Fig. 3B). The mRNA expression levels of HIF-1α and VEGF were also analyzed (Fig. 3C and D). The mRNA expression levels of VEGF were concordant with its protein expression levels (Fig. 3C). Notably, no significant differences in the mRNA expression levels of HIF-1α were observed between the negative control group and the rhDKK-1 treatment group (Fig. 3D). These results suggest that DKK-1 may increase HIF-1α protein translation or decrease HIF-1α degradation, rather than upregulate the transcription levels of HIF-1α. The nuclear localization of HIF-1α was also investigated. As shown in Fig. 3E and F, rhDKK-1 significantly increased HIF-1α nuclear translocation, as compared with the untreated group. These results indicate that HIF-1α translocates to the nucleus following DKK-1 exposure, and mediates DKK-1-induced VEGF expression.


Dickkopf‑related protein 1 induces angiogenesis by upregulating vascular endothelial growth factor in the synovial fibroblasts of patients with temporomandibular joint disorders.

Jiang SJ, Li W, Li YJ, Fang W, Long X - Mol Med Rep (2015)

Hypoxia inducible factor-1α (HIF-1α) mediates dickkopf-related protein 1 (DKK-1) regulation of vascular endothelial growth factor (VEGF) expression. (A) Western blotting demonstrated that the protein expression levels of HIF-1α in the synovial fibroblasts transiently transfected with scramble or HIF-1α small interfering (si)RNA 1 and 2. The proteins were extracted 72 h post-transfection. (B) HIF-1α siRNA (HIF-1α siRNA 2 as a target sequence) significantly inhibited the upregulatory effects of recombinant human (rh)DKK-1 on VEGF protein expression in synovial fibroblasts, as demonstrated by western blot analysis. (C and D) HIF-1α siRNA also inhibited the upregulatory effects of rhDKK-1 on the mRNA expression levels of VEGF in synovial fibroblasts. The mRNA expression levels of HIF-1α exhbited no significant changes between the control and rhDKK-1 treatment group. The results are presented as the mean ± standard error of the mean. **P<0.01, and ***P<0.001; one-way analysis of variance. RNA was extracted 48 h post-transfection. (E) Synovial fibroblasts treated with 10 ng/ml rhDKK-1 for 24 h exhibited HIF-1α nuclear translocation, as determined by immunofluorescence. Scale bars, 25 µm. (F) Quantification of nuclear translocation of HIF-1α in the vehicle group and rhDKK-1-treatment group. The results are presented as the mean ± standar error of the mean, **P<0.01; Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581813&req=5

f3-mmr-12-04-4959: Hypoxia inducible factor-1α (HIF-1α) mediates dickkopf-related protein 1 (DKK-1) regulation of vascular endothelial growth factor (VEGF) expression. (A) Western blotting demonstrated that the protein expression levels of HIF-1α in the synovial fibroblasts transiently transfected with scramble or HIF-1α small interfering (si)RNA 1 and 2. The proteins were extracted 72 h post-transfection. (B) HIF-1α siRNA (HIF-1α siRNA 2 as a target sequence) significantly inhibited the upregulatory effects of recombinant human (rh)DKK-1 on VEGF protein expression in synovial fibroblasts, as demonstrated by western blot analysis. (C and D) HIF-1α siRNA also inhibited the upregulatory effects of rhDKK-1 on the mRNA expression levels of VEGF in synovial fibroblasts. The mRNA expression levels of HIF-1α exhbited no significant changes between the control and rhDKK-1 treatment group. The results are presented as the mean ± standard error of the mean. **P<0.01, and ***P<0.001; one-way analysis of variance. RNA was extracted 48 h post-transfection. (E) Synovial fibroblasts treated with 10 ng/ml rhDKK-1 for 24 h exhibited HIF-1α nuclear translocation, as determined by immunofluorescence. Scale bars, 25 µm. (F) Quantification of nuclear translocation of HIF-1α in the vehicle group and rhDKK-1-treatment group. The results are presented as the mean ± standar error of the mean, **P<0.01; Student's t-test.
Mentions: To investigate the mechanisms underlying DKK-1 regulation of VEGF expression, the expression levels of HIF-1α, an upstream inducer of VEGF, were analyzed. HIF-1α siRNA 2 markedly inhibited the expression of HIF-1α (Fig. 3A). HIF-1α siRNA 2 was therefore chosen as the target sequence. As shown in Fig. 3B, the protein expression levels of HIF-1α were increased in synovial fibroblasts treated with rhDKK-1, as compared with the negative control. The protein expression levels of VEGF exhibited similar results. However, the upregulatory effects of rhDKK-1 on VEGF and HIF-1α protein expression were inhibited by HIF-1α siRNA (Fig. 3B). The mRNA expression levels of HIF-1α and VEGF were also analyzed (Fig. 3C and D). The mRNA expression levels of VEGF were concordant with its protein expression levels (Fig. 3C). Notably, no significant differences in the mRNA expression levels of HIF-1α were observed between the negative control group and the rhDKK-1 treatment group (Fig. 3D). These results suggest that DKK-1 may increase HIF-1α protein translation or decrease HIF-1α degradation, rather than upregulate the transcription levels of HIF-1α. The nuclear localization of HIF-1α was also investigated. As shown in Fig. 3E and F, rhDKK-1 significantly increased HIF-1α nuclear translocation, as compared with the untreated group. These results indicate that HIF-1α translocates to the nucleus following DKK-1 exposure, and mediates DKK-1-induced VEGF expression.

Bottom Line: The present study also investigated the effects of DKK‑1 on the migration of human umbilical vein endothelial cells (HUVEC).In addition, DKK‑1 induced HUVEC migration, and HIF‑1α siRNA inhibited DKK‑1‑induced cell migration.Furthermore, HIF‑1α may be associated with DKK‑1‑induced HUVEC activation.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‑MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079, P.R. China.

ABSTRACT
Angiogenesis has an important role in the progression of temporomandibular joint disorders (TMD). The aim of the present study was to explore the association between dickkopf‑related protein 1 (DKK‑1) and angiogenesis in TMD. The expression levels of DKK‑1 and vascular endothelial growth factor (VEGF) were quantified by an ELISA assay of the synovial fluid from patients with TMD. The correlation between DKK‑1 and VEGF was analyzed by Pearson correlation test. Synovial fibroblasts were isolated from patients with TMD and were subsequently treated with recombinant human DKK‑1, anti‑DKK‑1 antibody, hypoxia inducible factor‑1α (HIF‑1α), or small interfering RNA (siRNA). The expression levels of DKK‑1, HIF‑1α, and VEGF were subsequently quantified. The present study also investigated the effects of DKK‑1 on the migration of human umbilical vein endothelial cells (HUVEC). Increased expression levels of DKK‑1 were concordant with increased expression levels of VEGF in the synovial fluid from patients with TMD. In the synovial fibroblasts, DKK‑1 increased the expression levels of VEGF, and promoted HIF‑1α nuclear localization. In addition, DKK‑1 induced HUVEC migration, and HIF‑1α siRNA inhibited DKK‑1‑induced cell migration. The results of the present study indicate that DKK‑1 is associated with angiogenesis in the synovial fluid of patients with TMD. Furthermore, HIF‑1α may be associated with DKK‑1‑induced HUVEC activation.

Show MeSH
Related in: MedlinePlus