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Dynamics and gene expression of growth factor receptors in human cultured skin cells: Effects of UV radiation and calcium on EGF- and PDGF-receptors
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Growth factors transmit biological signals for the stimulation of cell growth and their stimulation may be involved in turnourigenesis. It is therefore of great importance to understand growth factor receptor reactions in response to stimuli such as calcium depletion or ultraviolet radiation, which normal human cells are invariably exposed to during their growth cycle. We have studied human skin cells i.e. fibroblasts, lceratinocytes and melanocytes and their growth factor receptor expression on the surface of cells, reactions in the plane of the cell membrane, intracellular trafficlcing, and gene expression after exposure to their ligand, serum, UVB radiation and calcium depletion. We have used Fluorescence recovery after photo bleaching (FRAP) to assess receptor characteristics in cell membranes, confocal laser scanning microscopy to visualize receptor internalization, Ratio imaging for calcium studies, Northern blot for detection of the gene for the epidermal growth factor receptor (EGF-R) and flow cytometry for cell surface receptor determination. Platelet-derived growth factor receptors (PDGF-R) and EGF-R were studied since they affect cell proliferation and viability.

Our results showed that the addition of PDGF increased receptor mobility characteristics in normal fibroblasts, also "starvation" of cells increased their receptor mobility. We could show that changes in both intra-and extracellular free Ca2+ influence the mobility characteristics of PDGF-ß2 receptors. We have demonstrated that the three celltypes display different basal EGF-R mobility characteristics. After UVB irradiation mobility characteristics increased in all cell types but with differences in diffusion coefficients or mobile fractions. Addition of antioxidant enzymes, catalase and superoxide dismutase, prior to UVB irradiated cells abolished the UY-induced receptor mobility changes. We have shown that a single physiologic dose ofUVB radiation alters the intracellular EGF-R distribution and intracellular transport in melanocytes. It also significantly alters the melanocyte phenotype. We were able to detect a constitutive EGF-R gene expression and showed that UVB-radiation induces a time-dependent induction in EGF-R mRNA in melanocytes. Human melanocytes express EGF-R on their cell surface and UVR induces time dependent changes in the number of receptors but the number of receptors does not correlate with the level of UV-induced EGF-R gene expression.

It is concluded that UV-radiation, growth factors and calcium, ubiquitous constituents of every day life, all having tremendous effects in vivo, also affect human cells in vitro in parameters studied that are of importance for proliferation, survival and tumourigenesis.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 88 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 674
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25680Local ID: 10056ISBN: 91-7219-965-2 (print)OAI: oai:DiVA.org:liu-25680DiVA: diva2:246228
Public defence
2001-05-18, Administrationshusets Aula, Universitetssjukhuset, Linköping, 09:30 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-06Bibliographically approved
List of papers
1. Lateral diffusion of PDGF β-receptors in human fibroblasts
Open this publication in new window or tab >>Lateral diffusion of PDGF β-receptors in human fibroblasts
1991 (English)In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 11, no 1, 43-52 p.Article in journal (Refereed) Published
Abstract [en]

When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum of PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding 'normal' and 'starved' cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0 x 10-10 cm2s-1 and >90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor. and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81072 (URN)10.1007/BF01118604 (DOI)
Available from: 2012-09-06 Created: 2012-09-06 Last updated: 2017-12-07Bibliographically approved
2. Both Intra- and Extracellular Ca2 Participate in the Regulation of the Lateral Diffusion of the PDGF-β2 Receptor
Open this publication in new window or tab >>Both Intra- and Extracellular Ca2 Participate in the Regulation of the Lateral Diffusion of the PDGF-β2 Receptor
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2000 (English)In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 20, no 2, 119-127 p.Article in journal (Refereed) Published
Abstract [en]

When the receptors for platelet-derived growth factor (PDGF) are activatedthey aggregate, become tyrosine-phosphorylated and elicit a cascade ofdown-stream signals, including mobilization of Ca2+ from intra- andextracellular stores. Receptor mobility in the plane of the membrane isa prerequisite for receptor aggregation and further signalling. Using humanforeskin fibroblasts (AG 1523) and fluorescence recovery afterphotobleaching (FRAP), we therefore assessed the lateral mobilitycharacteristics of PDGF-β2 receptors by their diffusioncoefficient (D), and fraction of mobile receptors (R). This was done oncells stimulated with either normal human serum (NHS) or PDGF underdifferent Ca2+-conditions.

The results suggest that both intra- and extracellular free Ca2+influence the mobility characteristics of the PDGF-β2receptor. Interestingly, the extracellular Ca2+ seems to imposegeneral restrictions on the mobility of receptors, since R increased whenextracellular Ca2+ was quenched with EGTA, whereas intracellularclamping of Ca2+ transients with MABTAM (BAPT/AM) primarily affectedD. When both intra- and extracellular Ca2+ were quenced, D remainedlow and R high, further supporting the proposition that they achievedistinct effects. Inhibition of tyrosine phosphorylation with Erbstatin,partly inhibited the NHS effects and released PDGF-induced receptorimmobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGFinduced changes in intracellular free [Ca2+]. In view of the presentdata it might have important effects on the state of the receptor in themembrane, for instance by regulating its lateral mobility, communicationwith other receptors and signalling functions in the membrane.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25447 (URN)10.1023/A:1005519501194 (DOI)9893 (Local ID)9893 (Archive number)9893 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-09-06Bibliographically approved
3. UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts
Open this publication in new window or tab >>UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts
1996 (English)In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 16, no 3, 227-238 p.Article in journal (Refereed) Published
Abstract [en]

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz, with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2 ± 0.2 x 10-10 cm2/s, and 1.8 ± 0.2 x 10-10 cm2/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3 ± 0.3 x 10-10 cm2/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1 ± 0.8 x 10-10 cm2/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81073 (URN)10.1007/BF01207337 (DOI)
Available from: 2012-09-06 Created: 2012-09-06 Last updated: 2017-12-07Bibliographically approved
4. UVB radiation increases EGF receptor mobility and trafficking in human melanocytes
Open this publication in new window or tab >>UVB radiation increases EGF receptor mobility and trafficking in human melanocytes
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

For the human skin, UVB-radiation (290-320nm) is a very potent injurious agent. UV radiation is absorbed in the epidermis and reaching the melanocytes leads to proliferation via activation of growth factor reccptors. This may play a key role in the clonal expansion of melanocytes and be a critical step in carcinogenesis. We show that UVB-irradiated human epidermal melanocytes display an increased mobility of epidermal growth factor receptors (EGF-R) in the plane of the cell membrane, and that UVB affects the intracellular EGF-R transport to the nucleus. Using fluorescence photobleaching technique we show a time and dose dependent increase in the diffusion coefficient and mobile fraction of EGF-R. EGF-Rdiffuse with a low rate within the cell membrane in control cells, and the mobility increases 4-fold after single physiologic doses of UVB. Three-dimensional confocal microscopy reveals that EGF-R display a strilting difference in receptor distribution and intracellular transport before and after UVB irradiation. The EGF-Rclearly eo-localize with clathrin-coated pits within the cells. These results indicate that already single physiologic doses of UVB affect growth factor receptor mobility in cell membranes and intracellular trafficlting. This may be an important early step in the ultraviolet radiation-induced signal transduction pathway leading to cell proliferation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81074 (URN)
Available from: 2012-09-06 Created: 2012-09-06 Last updated: 2012-09-06Bibliographically approved
5. UVB radiation increases EGF-R gene expression in cultured human normal melanocytes
Open this publication in new window or tab >>UVB radiation increases EGF-R gene expression in cultured human normal melanocytes
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Epidermal growth factor (EGF) and its receptor are thought to play important roles in the UV response of mammalian cells. Since UVB radiation has been shown to influence melanocyte growth and development, we sought to determine whether UVB would alter the expression of the gene for EGF-R. Using Northern blot, the present study exanlined the expression of EGF-R mRNA in cultured normal human melanocytes exposed to a single physiologic dose of UVB radiation. Total cellular RNA was extracted from cultured normal human melanocytes at various time-points after irradiation or sham-irradiation. Constitutive expression ofEGF-Rmfu'!A was detected in the melanocytes. After UVB irradiation with a single dose of 6o or 180 mJ/cm2, the EGF-R rnRNA decreased within 12 h to approximately 50 % of the initial value. After 24 h there was a subsequent increase and a peak at 36 h, showing a three-fold increase. After 48 hours a decline was seen, but the expression of EGF-R mRNA was still doubled as compared to control.

Using flow cytometric analysis EGF-R on melanocytes were detected. The median EGF-R immunofluorescence was decreased after UVB-irradiation as compared to non-irradiated cells. The lowest EGF-R immunofluorescence was seen 12 hours after UVB irradiation. Witi1in 48 hours post irradiation we could not detect any increase in EGF-R that we would correlate to the increased EGFR gene expression seen at 36 hours. We conclude ti1at the steady-state levels of EGF-R mRNAis upregulated by single exposure to UVB.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81075 (URN)
Available from: 2012-09-06 Created: 2012-09-06 Last updated: 2012-09-06Bibliographically approved

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