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Enhanced keratinocyte proliferation and migration in co-culture with fibroblasts.

Wang Z, Wang Y, Farhangfar F, Zimmer M, Zhang Y - PLoS ONE (2012)

Bottom Line: These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition.Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation.These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic and Aesthetic Surgery, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China.

ABSTRACT
Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11(th) to 15(th) day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

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Effects of different cytokines on the growth of Keratinocytes.Keratinocytes and fibroblasts were respectively stained with PHK26 and PHK2 before co-culture. Keratinocytes were cultured with fibroblasts (K/F), or with 10 ng/ml, 20 ng/ml and 40 ng/ml of HB EGF (K h10, K h20 and Kh40), IL-1α (Ki10, K i20 and K i40) or TGFβ1 (K t10, K t20 and K t40), or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 10 ml after harvested on day 5 following the culture. Four cultures in each condition. Keratinocytes were counted by Trypan Blue exclusion and Flow Cytometry.
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pone-0040951-g004: Effects of different cytokines on the growth of Keratinocytes.Keratinocytes and fibroblasts were respectively stained with PHK26 and PHK2 before co-culture. Keratinocytes were cultured with fibroblasts (K/F), or with 10 ng/ml, 20 ng/ml and 40 ng/ml of HB EGF (K h10, K h20 and Kh40), IL-1α (Ki10, K i20 and K i40) or TGFβ1 (K t10, K t20 and K t40), or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 10 ml after harvested on day 5 following the culture. Four cultures in each condition. Keratinocytes were counted by Trypan Blue exclusion and Flow Cytometry.

Mentions: Since the fibroblasts did not produce significant levels of HB-EGF in the experiments described above (Fig. 3A), IL-1α and TGFβ1 or other cytokines most likely play a critical role in fibroblast-stimulated keratinocyte proliferation. However, the levels of IL-1α and TGFβ1 where the cells were co-cultured in the transwell were similar to the levels when the cells were cultured in direct contact with each other. Moreover, the stronger keratinocyte proliferation induced in co-cultures required direct contact between keratinocytes and fibroblasts. To further determine the roles of these two cytokines as well as the effect of cell-to-cell contact on keratinocyte proliferation, keratinocytes were cultured in the presence of different concentrations of HB-EGF, IL-1α and TGFβ1 and then compared to the keratinocytes/fibroblasts co-culture. Fig. 4 shows that all three cytokines stimulated kerotinocyte proliferation in a dose-dependent manner, but the level of cytokine required to reach the same level of growth stimulation found in the co-cultures was as much as 10 to 20-fold higher (15–30 ng/ml) than what was found in the co-culture media (1–2 ng/ml). Therefore, it is possible that the local concentrations of these cytokines in the cell-to-cell contact micro-environment are much higher than the overall levels in the media, which may be sufficient to affect proliferation.


Enhanced keratinocyte proliferation and migration in co-culture with fibroblasts.

Wang Z, Wang Y, Farhangfar F, Zimmer M, Zhang Y - PLoS ONE (2012)

Effects of different cytokines on the growth of Keratinocytes.Keratinocytes and fibroblasts were respectively stained with PHK26 and PHK2 before co-culture. Keratinocytes were cultured with fibroblasts (K/F), or with 10 ng/ml, 20 ng/ml and 40 ng/ml of HB EGF (K h10, K h20 and Kh40), IL-1α (Ki10, K i20 and K i40) or TGFβ1 (K t10, K t20 and K t40), or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 10 ml after harvested on day 5 following the culture. Four cultures in each condition. Keratinocytes were counted by Trypan Blue exclusion and Flow Cytometry.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401236&req=5

pone-0040951-g004: Effects of different cytokines on the growth of Keratinocytes.Keratinocytes and fibroblasts were respectively stained with PHK26 and PHK2 before co-culture. Keratinocytes were cultured with fibroblasts (K/F), or with 10 ng/ml, 20 ng/ml and 40 ng/ml of HB EGF (K h10, K h20 and Kh40), IL-1α (Ki10, K i20 and K i40) or TGFβ1 (K t10, K t20 and K t40), or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 10 ml after harvested on day 5 following the culture. Four cultures in each condition. Keratinocytes were counted by Trypan Blue exclusion and Flow Cytometry.
Mentions: Since the fibroblasts did not produce significant levels of HB-EGF in the experiments described above (Fig. 3A), IL-1α and TGFβ1 or other cytokines most likely play a critical role in fibroblast-stimulated keratinocyte proliferation. However, the levels of IL-1α and TGFβ1 where the cells were co-cultured in the transwell were similar to the levels when the cells were cultured in direct contact with each other. Moreover, the stronger keratinocyte proliferation induced in co-cultures required direct contact between keratinocytes and fibroblasts. To further determine the roles of these two cytokines as well as the effect of cell-to-cell contact on keratinocyte proliferation, keratinocytes were cultured in the presence of different concentrations of HB-EGF, IL-1α and TGFβ1 and then compared to the keratinocytes/fibroblasts co-culture. Fig. 4 shows that all three cytokines stimulated kerotinocyte proliferation in a dose-dependent manner, but the level of cytokine required to reach the same level of growth stimulation found in the co-cultures was as much as 10 to 20-fold higher (15–30 ng/ml) than what was found in the co-culture media (1–2 ng/ml). Therefore, it is possible that the local concentrations of these cytokines in the cell-to-cell contact micro-environment are much higher than the overall levels in the media, which may be sufficient to affect proliferation.

Bottom Line: These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition.Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation.These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic and Aesthetic Surgery, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China.

ABSTRACT
Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11(th) to 15(th) day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

Show MeSH