The effects of TGF-alpha, IL-1beta and PDGF on fibroblast adhesion to ECM-derived matrix and KGF gene expression

Abstract

The goal of this study was to elucidate the control mechanisms by which exogenous proteins regulate keratinocyte growth factor (KGF) expression in fibroblasts adhered to differing substrates and thereby provide insights into both fundamental in vitro cell signaling and cell-biomaterial interaction research. A serum-free culture system in which cells maintained their proliferative capacity was established and employed. The addition of transforming growth factor- alpha (TGF-alpha), interleukin-1beta (IL-1beta) and platelet-derived growth factor-BB (PDGF-BB) individually showed no effect on KGF protein release, however, IL-1beta addition led to increased KGF mRNA transcription, intracellular KGF protein synthesis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) release. Intracellular KGF protein synthesis and extracellular release were enhanced when fibroblasts were treated with a combination of IL-1beta and PDGF-BB which suggests KGF synthesis and release are largely regulated by synergistic mechanisms. Surface-bound fibronectin-derived ligands and individual exogenous proteins promoted fibroblast adhesion to semi-interpenetrating polymer networks (sIPNs) but did not stimulate KGF release despite enhancement of KGF mRNA transcription. Additionally, serum conditioning was found to have a significant impact on KGF synthesis and the subsequent mechanisms controlling KGF release. This study demonstrates that KGF release from fibroblasts is likely regulated by multiple mechanisms involving post-transcriptional and exocytic controls which may be impacted by the presence of serum and how serum is removed from the in vitro cell environment.

Figure 1

Adherent human dermal fibroblast density on TCPS with or without serial transitioning (ST). Cells were split into two groups. One group of cells were passaged directly into DMEM/F12 with or without 5% FBS. The other group of cells were serially passaged into FBM or DMEM/F12 with or without 5% FBS by gradually reducing serum concentration. Cell seeding density was 10,000 cells/cm². Adherent cell density was quantified by LIVE/DEAD assay at 24 (), 72 (), 120 () and 168 (□) hours. Complete growth medium FGM-2 served as a control. All data presented as average ± standard deviation (n=3). Dashed line represents the cell confluency. §: Significantly different compared to cells without transitioning at the same time point, p<0.05.

Figure 2

KGF release from fibroblasts cultured on TCPS in FBM (), FBM+2% FBS (), FBM+5%FBS (), and FGM-2 (□). Serially transitioned cells were cultured on TCPS in serum-free and serum-containing media. FGM-2 served as a positive control using cells which did not undergo serial transitioning. KGF concentrations in cell culture supernatant from 24 to 168 hours were measured by ELISA. All data presented as average ± standard deviation (n=3). ‡: Significantly different compared to FBM+2%FBS, FBM+5%FBS, and FGM-2 at the same time point, p < 0.05. §: Significantly different compared to FBM+5%FBS, and FGM-2 at the same time point, p < 0.05. ¤: Significantly different compared to FGM-2 at the same time point, p < 0.05.

Figure 3

Adherent cell density of fibroblasts cultured on unmodified sIPN (), GGG-modified sIPN (), RGD-modified sIPN () and TCPS (□) at 24, 72, 120 and 168 hours in FBM supplemented with no exogenous protein (A), 10ng/mL TGF-α (B), 10ng/mL IL-1β (C), and 10ng/mL PDGF-BB (D). Cells were stained with LIVE/DEAD fluorescent assay kit and observed under microscope at 10× magnification. Images were recorded for cell counting. Five images per sample were taken at random fields of view. All data presented as average ± standard deviation (n=3). ‡: Significantly different compared to unmodified-sIPN with treatment by the same exogenous protein, p < 0.05. §: Significantly different compared to GGG-sIPN with treatment by the same exogenous protein, p < 0.05. N/A: Data not collected for that time point.

Figure 4

Inverted microscope images of human dermal fibroblasts adherent to RGD-modified (A–G) and TCPS (H) surfaces at 24 (A–D, H) and 168 hours (E–G). Cells were cultured in FBM (D, H) or FBM supplemented with TGF-α (A, E), IL-1β (B, F) and PDGF-BB (C, G). Cells were stained with LIVE/DEAD fluorescent assay kit and incubated at 37°C for 30 minutes followed by observation under inverted microscope. Live and dead cells were stained by calcium-AM (green) and ethidium homodimer (red) respectively. The sIPN was slightly stained red in the background. All images were taken at 10× magnification. Scale bar represents 100 μm.

Figure 5

GM-CSF release in IL-1β (10ng/mL) supplemented FBM from human dermal fibroblasts adhered to GGG-modified sIPN (), RGD-modified sIPN (), unmodified gelatin sIPN () and TCPS (□). represents the GM-CSF release from fibroblasts cultured on TCPS in FBM without supplemented IL-1β. All data presented as average ± standard deviation (n=3). §: Significantly different compared to unmodified sIPN, p<0.05. ‡: Significantly different compared to RGD modified sIPN, p<0.05;

Figure 6

KGF mRNA levels of human dermal fibroblasts cultured in FBM (), FBM+5%FBS () and FBM+10ng/mL IL-1β (□) on TCPS; in FBM () and FBM+10ng/mL IL-1β () on unmodified sIPN as well as in FBM on RGD-modified sIPN () at 24, 72 and 120 hours presented as the ratio between KGF and RPII band intensities. KGF and RPII band intensities were measured by ImageJ software version 1.32j. FBM+5%FBS served as a control. All data presented as average ± standard deviation (n=3 for TCPS and RGD-modified sIPN samples; n=2 for unmodified sIPN samples). N/A: data not collected for that time point.

Figure 7

KGF protein levels in the lysate of human dermal fibroblasts cultured on TCPS in different media: FBM without serial transitioning (), FBM (), FBM+5% FBS (), FBM+10ng/mL IL-1β (), FGM-2 () and FBM+10ng/mL IL-1β+10ng/mL PDGF-BB (□). FBM+5%FBS and FGM-2 served as controls. Cells were seeded in 6-well plates and lysed by M-PER with protease inhibitor at 48 and 120 hours. The KGF concentration in cell lysate was measured by ELISA and the total protein concentration was quantified by a BCA assay. The KGF protein expression was presented as the ratio of KGF to total cell protein. All data presented as average ± standard deviation (n=3). ‡: compared to FBM with adaptation at the same time point, p<0.05; §: compared to the same culture medium at 48 hours, p<0.05. N/A: Data not collected for that media.

Figure 8

KGF protein concentrations in the lysate of human dermal fibroblasts cultured on unmodified sIPN with FBM () and with FBM+10ng/mL IL-1β () as well as on RGD-modified sIPN with FBM (□). Cells were seeded onto sIPNs which were fitted into 48-well plates. Cells were then lysed by M-PER with protease inhibitor at 24 and 72 hours. KGF concentrations were measured by ELISA. All data presented as average ± standard deviation. §: significantly different compared to unmodified sIPN when cells were cultured in FBM, p<0.05. *: sample size n=2, otherwise n=3.