Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors
2000; Elsevier BV; Volume: 275; Issue: 25 Linguagem: Inglês
10.1074/jbc.m000008200
ISSN1083-351X
AutoresHidenori Shiraha, Kiran Gupta, Kathryn Drabik, Alan Wells,
Tópico(s)Skin Protection and Aging
ResumoWound healing is compromised in aging adults in part due to decreased responsiveness of fibroblasts to extracellular signals. However, the cellular mechanisms underlying this phenomenon are not known. Aged dermal fibroblasts with reduced remaining replicative capacities demonstrated decreased epidermal growth factor (EGF)-induced cell migrative and cell proliferative capacities, as reported previously. Thus, as cells approach senescence, programmedin vivo or in vitro, EGF responsiveness is preferentially lost. To define the rate-limiting signaling event, we found that the activity of two different EGF receptor (EGFR)-signaling pathways to cell migration (phospholipase-C γ) and/or mitogenesis (extracellular signal/regulated-mitogen-activated kinases) were decreased in near senescent cells despite unchanged levels of effector molecules. Aged cells presented decreased levels of EGFR, although insulin receptor and transferrin receptor levels were relatively unchanged. EGFR mRNA levels and production of new transcripts decreased during aging, suggesting that this preferential loss of EGFR was due to diminished production, which more than counteracts the reduced ligand-induced receptor loss. Since these data suggested that the decrement in EGF was rate-limiting, higher levels of EGFR were established in near senescent cells by electroporation of EGFR cDNA. These cells presented higher levels of EGFR and recovered their EGF-induced migration and proliferation responsiveness. Thus, the defect in EGF responsiveness of aged dermal fibroblasts is secondary to reduced EGFR message transcription. Our experimental model suggests that EGFR gene delivery might be an effective future therapy for compromised wound healing. Wound healing is compromised in aging adults in part due to decreased responsiveness of fibroblasts to extracellular signals. However, the cellular mechanisms underlying this phenomenon are not known. Aged dermal fibroblasts with reduced remaining replicative capacities demonstrated decreased epidermal growth factor (EGF)-induced cell migrative and cell proliferative capacities, as reported previously. Thus, as cells approach senescence, programmedin vivo or in vitro, EGF responsiveness is preferentially lost. To define the rate-limiting signaling event, we found that the activity of two different EGF receptor (EGFR)-signaling pathways to cell migration (phospholipase-C γ) and/or mitogenesis (extracellular signal/regulated-mitogen-activated kinases) were decreased in near senescent cells despite unchanged levels of effector molecules. Aged cells presented decreased levels of EGFR, although insulin receptor and transferrin receptor levels were relatively unchanged. EGFR mRNA levels and production of new transcripts decreased during aging, suggesting that this preferential loss of EGFR was due to diminished production, which more than counteracts the reduced ligand-induced receptor loss. Since these data suggested that the decrement in EGF was rate-limiting, higher levels of EGFR were established in near senescent cells by electroporation of EGFR cDNA. These cells presented higher levels of EGFR and recovered their EGF-induced migration and proliferation responsiveness. Thus, the defect in EGF responsiveness of aged dermal fibroblasts is secondary to reduced EGFR message transcription. Our experimental model suggests that EGFR gene delivery might be an effective future therapy for compromised wound healing. epidermal growth factor receptor glyceraldehyde-3-phosphate dehydrogenase green fluorescence protein mitogen-activated protein kinase population doublings remaining phospholipase C-γ extracellular signal-regulated kinase Dulbecco's modified Eagle's medium fetal bovine serum polyacrylamide gel electrophoresis bromodeoxyuridine Problems in wound and skin repair constitute major medical problems for aging adults. The age-related loss of wound healing capacity leads to a high risk of surgical wound dehiscence and infection (1.Lober C.W. Fenske N.A. South. Med. J. 1991; 84: 1444-1446Crossref PubMed Scopus (22) Google Scholar, 2.Nicolle L.E. Huchcroft S.A. Cruse P.J. J. Clin. Epidemiol. 1992; 45: 357-364Abstract Full Text PDF PubMed Scopus (27) Google Scholar). All phases of wound healing are diminished (3.Ashcroft G.S. Horan M.A. Ferguson M.W. J. Anat. 1995; 187: 1-26PubMed Google Scholar, 4.Gerstein A.D. Phillips T.J. Rogers G.S. Gilchrest B.A. Dermatol. Clin. 1993; 11: 749-757Abstract Full Text PDF PubMed Google Scholar). In normal wound healing, fibroblasts are recruited from the surrounding intact tissue into the granulation tissue to proliferate and regenerate a new dermal layer in response to various factors presented in the wound fluid (5.Hay E.D. Curr. Opin. Cell Biol. 1993; 5: 1029-1035Crossref PubMed Scopus (138) Google Scholar). Thus, both fibroblast motility and mitogenesis are critical for wound repair. During aging dermal fibroblasts lose both proliferative and basal migrative capacity (3.Ashcroft G.S. Horan M.A. Ferguson M.W. J. Anat. 1995; 187: 1-26PubMed Google Scholar); this seems to be a major reason for compromised wound healing in aged adults. To compensate, growth factors have been used as adjuvants in non-healing wounds with limited success (6.Beck L.S. DeGuzman L. Lee W.P. Xu Y. Siegel M.W. Amento E.P. 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Dermatol. 1994; 12: 157-169Abstract Full Text PDF PubMed Scopus (170) Google Scholar); among those that are most robust are factors that activate the epidermal growth factor receptor (EGFR)1 (12.Barrandon Y. Green H. Cell. 1987; 50: 1131-1137Abstract Full Text PDF PubMed Scopus (568) Google Scholar, 13.Blay J. Brown K.D. J. Cell. Physiol. 1985; 124: 107-112Crossref PubMed Scopus (189) Google Scholar, 14.Carpenter G. Cohen S. Natl. Cancer Inst. Monogr. 1978; 48: 149-156Google Scholar, 15.Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar, 16.Gospodarowicz D. Mescher A.L. J. Cell. Physiol. 1977; 93: 117-127Crossref PubMed Scopus (79) Google Scholar). These factors, transforming growth factor-α and heparin-binding EGF-like growth factor in particular (17.Blotnick S. Peoples G.E. Freeman M.R. Eberlein T.J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2890-2894Crossref PubMed Scopus (279) Google Scholar, 18.Kiritsy C.P. Lynch A.B. Lynch S.E. Crit. Rev. Oral Biol. Med. 1993; 4: 729-760Crossref PubMed Scopus (160) Google Scholar, 19.Steenfos H.H. Scand. J. Plast. Reconstr. Surg. Hand Surg. 1994; 28: 95-105Crossref PubMed Scopus (171) Google Scholar), are present during all stages of wound repair, suggesting that they play important roles in orchestrating wound repair. EGFR levels on dermal fibroblasts have been seen to decline in aging, with this decline correlating with decreased mitogenic responsiveness to EGF (20.Liu Y. Guyton K.Z. Gorospe M. Xu Q. Kokkonen G.C. Mock Y.D. Roth G.S. Holbrook N.J. J. Biol. Chem. 1996; 271: 3604-3607Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 21.Reenstra W.R. Yaar M. Gilchrest B.A. Exp. Cell Res. 1996; 227: 252-255Crossref PubMed Scopus (42) Google Scholar). The other critical cell property induced by EGFR signaling, cell motility, has not been investigated during aging. It has been reported that endothelial cell proliferative and migratory responses to a different growth factor, fibroblast growth factor, decrease as human umbilical vein endothelial cell senesce (22.Garfinkel S. Hu X. Prudovsky I.A. McMahon G.A. Kapnik E.M. McDowell S.D. Maciag T. J. Cell Biol. 1996; 134: 783-791Crossref PubMed Scopus (74) Google Scholar). However, since fibroblasts did not demonstrate a similar correlation between aging and fibroblast growth factor nonresponsiveness (23.Garfinkel S. Wessendorf J.H. Hu X. Maciag T. Biochim. Biophys. Acta. 1996; 1314: 109-119Crossref PubMed Scopus (12) Google Scholar) and human umbilical vein endothelial cells are dependent on fibroblast growth factor for growth, the causal nature of this correlation between senescence and responsiveness to growth factors is uncertain and remains to be demonstrated. Furthermore, the crucial question of whether this concomitant decline in EGFR levels and cell responsiveness is causally or only coincidentally related to a global cellular decline in functioning remains unknown. Recent advances in signal transduction research have defined intracellular signaling pathways that are required for both motility and mitogenesis. Full EGF-induced cell migration requires phospholipase-C γ (PLCγ) signaling (24.Chen P. Xie H. Sekar M.C. Gupta K. Wells A. J. Cell Biol. 1994; 127: 847-857Crossref PubMed Scopus (285) Google Scholar, 25.Ji Q.S. Ermini S. Baulida J. Sun F.L. Carpenter G. Mol. Biol. Cell. 1998; 9: 749-757Crossref PubMed Scopus (63) Google Scholar). Inhibition of PLCγ signaling specifically abrogates cell motility but not proliferation (24.Chen P. Xie H. Sekar M.C. Gupta K. Wells A. J. Cell Biol. 1994; 127: 847-857Crossref PubMed Scopus (285) Google Scholar, 26.Chen P. Xie H. Wells A. Mol. Biol. Cell. 1996; 7: 871-881Crossref PubMed Scopus (100) Google Scholar); thus, activation of this pathway could be thought of as an indicator of EGFR-mediated cell locomotion. Another major signaling pathway from EGFR is via mitogen-activated protein kinase (MAPK) signaling pathway, which is required for both cell mitogenesis and cell migration (27.Pages G. Lenormand P. L'Allemain G. Chambard J.C. Meloche J. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (925) Google Scholar, 28.Xie H. Pallero M.A. Gupta K. Chang P. Ware M.F. Witke W. Kwiatkowski D.J. Lauffenburger D.A. Murphy-Ullrich J.E. Wells A. J. Cell Sci. 1998; 111: 615-624Crossref PubMed Google Scholar); the point of divergence of these two cell responses occurs downstream of MAPK kinase in this pathway (28.Xie H. Pallero M.A. Gupta K. Chang P. Ware M.F. Witke W. Kwiatkowski D.J. Lauffenburger D.A. Murphy-Ullrich J.E. Wells A. J. Cell Sci. 1998; 111: 615-624Crossref PubMed Google Scholar) but remains to be deciphered. Despite the uncertainty of all the signals required for either motility or mitogenesis, these two intracellular effectors, PLCγ and Erk-MAPK, provide intracellular barometers of signal transduction and intermediary markers of cell locomotive and proliferative responses, respectively. We hypothesized that decreased EGFR expression causally results in impaired responsiveness of dermal fibroblasts. We measured negative effects of cell aging on EGF-induced cell migration and mitogenesis. By examining the activation status of the intermediary PLCγ and Erk-MAPK effectors, we determined that the diminished responsiveness was at least partly due to a receptor or immediate post-receptor defect. Examining production and consumption of EGFR demonstrated that the age-related decrease in mRNA transcription outweighed the reduced ligand-induced degradation. Furthermore, we assessed the effect of EGFR restoration in aged fibroblasts. Re-expression of EGFR to the levels seen in young fibroblasts restored the EGFR-mediated responses in the near senescent cells. These data hint at an age-related promoter element in the EGFR gene. Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3). Human diploid fibroblasts from 16-week female fetus(GM04522A, P6) and 19-year-old female (GM08399, P5) were purchased from the NIA cell repository (Camden, NJ). Cells were passaged by 1:8 split to increase cumulative cell population doubling level by 3 on each passage (29.Cristofalo V.J. Charpentier R. Phillips P.D. Celis J.E. Cell Biology: A Laboratory Handbook. Academic Press, Inc., San Diego1998: 313-318Google Scholar). Population doublings remaining (PDR) was back-calculated from passaging cells to senescence; PDR indicates the remaining replicative capacity, so that comparisons can be made between cells of different initial doubling levels. EGF was obtained from Collaborative Biomedical Products (Bedford, MA). EGF-induced proliferation was determined by incorporation of [3H]thymidine by standard procedures (24.Chen P. Xie H. Sekar M.C. Gupta K. Wells A. J. Cell Biol. 1994; 127: 847-857Crossref PubMed Scopus (285) Google Scholar). Cells were grown to confluence in 12-well plates and quiesced for 48 h in Dulbecco's modified Eagle's medium (DME) with 0.1% dialyzed FBS and then incubated with EGF (1 nm) for 16 h. [3H]Thymidine (5 μCi/well) was added, and cells were incubated for a further 10 h. Basal and EGF-induced migration was assessed by the ability of the cells to move into an acellular area as described previously (15.Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar). Cells were plated on a 6-well plastic dish and grown to confluence in DME with 7.5% FBS. After a 48-h quiescence in media with 0.1% dialyzed FBS, an area was denuded by a rubber policeman. The cells were then treated with or without EGF (1 nm; a concentration that provided maximal motility of Hs68 cells (data not shown)) and incubated at 37 °C. Photographs were taken at 0 h and 24 h, and the distance traveled by the cells at the acellular front was determined. The levels of target molecules were assessed by immunoblotting. Cells (4 × 106) were treated with EGF (1–10 nm). Cell lysates were separated on 7.5% or 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The blot was probed by anti-EGFR antibody (05–104, Upstate Biotechnology Incorporated, Lake Placid, NY), anti-β-insulin receptor antibody (I16630, Transduction Laboratories, Lexington, KY), anti-transferrin receptor antibody (GR09, Calbiochem), anti-α-actin antibody (A-2066, Sigma), anti-phospho-Erk-MAPK (9101, New England Biolabs, Beverly, MA), anti-pan MAPK antibody (9102, New England Biolabs), anti-PLCγ1 antibody (05-163, Upstate Biotechnology Inc.), or anti-phosphotyrosine antibody (PY-20, Transduction Laboratories). Target proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies followed by development with a colorimetric method (Promega, Madison, WI). The expression levels of EGFR, transferrin receptor, and β-insulin receptor were determined by densitometry (NIH Image) and reported as a ratio to α-actin, chosen as a housekeeping gene. The activation status of PLCγ was determined by immunoprecipitation followed by immunoblotting. Cells (2 × 107) were treated with EGF as described, and lysates were incubated overnight at 4 °C with anti-PLCγ1 antibody (05-163, Upstate Biotechnology Inc.). Immuno-complexes were captured with protein G-agarose beads and washed three times with 20 nm HEPES buffer, pH 7.4, containing 10% glycerol, 0.1% Triton X-100, 500 mm sodium chloride, 1 mm sodium vanadate. Immunoprecipitates were analyzed following immunoblotting using anti-phosphotyrosine antibody (PY-20, Transduction Laboratories). The expression level of EGFR was determined by a standard binding assay (30.Wiley H.S. Walsh B.J. Lund K.A. J. Biol. Chem. 1989; 264: 18912-18920Abstract Full Text PDF PubMed Google Scholar). Cells were grown to confluence in 12-well plates and washed twice with binding buffer (DME with 1% bovine serum albumin (Fraction V; Sigma)). 0.1 nm[125I]EGF (ICN, Irvine, CA) was added to unlabeled EGF (0–10 nm) in binding buffer. Plates were incubated for 2 h at 4 °C, and then the unbound-labeled EGF was collected. Cells were lysed with lysis buffer (Tris-buffered saline with 0.5% SDS). Both unbound and bound radioactivity was counted by γ-counter (Beckman Instruments). The number of binding sites was calculated by Scatchard analysis using linear regression. Northern blot analyses to quantitate message levels were performed using 3 μg of mRNA purified by TRIzol (Life Technologies, Inc.) and Oligo-(dT) cellulose (Life Technologies, Inc.). RNA was electrophoresed and transferred to nylon membrane Hybond N+ (Amersham Pharmacia Biotech) and probed according to the standard procedures. A probe for EGFR was prepared from human EGFR cDNA (15.Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar). A probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was prepared from the human GAPDH cDNA (ATCC). The probes were labeled with α[32P]-dCTP using the Ready To Go random primer labeling kit (Amersham Pharmacia Biotech). Blots were analyzed by phospho-image analyzer Molecular Imager System GS-525 and Molecular analyst (Bio-Rad). For RNA stability analysis, cells were treated with actinomycin D (Sigma) (5 μm) for 2.5 h before collecting RNA. P5 and P18 of Hs68 cells were treated with Nonidet P-40 lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2, 0.5% Nonidet P-40) for 10 min on ice. Intact nuclei were collected by centrifugation 500 × g for 5 min at 4 °C. Nuclei then were incubated with 32P-labeled UTP and unlabeled ATP, CTP, and GTP to label the nascent RNA. 32P-labeled RNAs were isolated by TRIzol (Life Technologies, Inc.) and hybridized with dot-blotted DNA on nylon membrane Hybond N+ (Amersham Pharmacia Biotech) for 12 h at 42 °C. The membranes were washed and exposed for autoradiography. The amounts of gene transcription were determined by densitometry. Cells were plated in 10-cm culture plate and grown to confluence in DME with 10% FBS. Cells were treated with interferon-γ-inducible protein-10 (50 ng/ml) (Peprotech, Rocky Hill, NJ) for 4 h or forskolin (25 μm) (Sigma) for 30 min. Ice-cold extraction buffer (50% ethanol, 0.1 nHCl) was added and incubated on ice for 15 min. Extracts were lyophilized and re-suspended in 100 μl of water. cAMP was quantitated using a cAMP assay kit (Amersham Pharmacia Biotech). After the extraction, cells were lysed with 0.1 n NaOH and analyzed for protein content using the Bradford protein assay. Near senescent CRL-7815 cells from an 83-year-old male donor (tested at P6; reproducibly senesced at P7) and Hs68 cells (P18, PDR3; reproducibly senesced at P19) were electroporated with a human EGFR cDNA driven from the SV-40 early promoter (15.Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar). Green fluorescence protein (GFP) plasmid (Life Technologies, Inc.) was introduced in parallel, and GFP expression was determined by fluorescence microscopy after 48 h to assess efficiency of electroporation. Approximately 107cells were electroporated (500 μF, 0.320 kV) with 20 μg of DNA in a total volume of 500 μl. Electroporated cells were incubated for 48 h in DME with 0.1% dialyzed FBS before experimentation. Cells were grown to confluence in 6-well plastic plates and washed twice with binding buffer (as described in Scatchard analyses for EGFR expression levels). Cells were pre-incubated with binding buffer for 1 h at 37 °C and incubated in 0.1 nm125I-EGF (ICN) for 10, 8, 6, 4, 2, and 0 min at 37 °C. Cells were washed with ice-cold binding buffer at the end of incubation. Surface bound125I-EGF was obtained by collecting two washes of the cells with acid strip buffer (50 mm glycine, 100 mmNaCl, 2 mg/ml polyvinlpyrrolidone, 2 m urea, pH 3.0 adjusted with HCl). Internalized 125I-EGF were obtained by lysing the cells with 1 m NaOH. Both surface and internalized radioactivity was counted by γ counter (Beckman). The endocytic rate constants were calculated by the time course of loss of surface-bound EGF and accumulation of internalized EGF (31.Wiley H.S. Cunningham D.D. Cell. 1981; 25: 433-440Abstract Full Text PDF PubMed Scopus (165) Google Scholar). Near senescent cells (Hs68 P18, PDR3) were electroporated with 20 μg of EGFR plasmid and 20 μg of GFP plasmid or mock electroporated without plasmid. These cells were mixed 1:1 and plated for analyses. Cells that express GFP are presumed to also express exogenous EGFR. The cells were incubated with Dulbecco's modified Eagle's medium with 0.1% dialyzed FBS for 48 h before EGF (1 nm) treatment. After a 16-h incubation with EGF, cells were labeled with 10 μm BrdUrd for 1 h. Cells were observed by fluorescent microscope, then cells were fixed by 70% ethanol and stained with a BrdUrd staining kit (HCS24, Oncogene research products, Cambridge, MA) Hs68 cells reproducibly senesce at P19 (n = 4). We note two separate populations in terms of EGF responsiveness: early- and mid-passage (>PDR10) and late-passage ( 470 μm/day (average 1.7-fold induction); thereafter it fell precipitously to 300 μm/day by P18 (PDR3) (1.1-fold induction). Basal thymidine incorporation remained low but steady during early- and mid-passage (average 8300 cpm) until late passage (P18, PDR3 presented 850 cpm) (Fig. 1 B). EGF-induced mitogenesis was strong early (15-fold for early- and mid-passage) but disappeared as cells approached senescence (0.99-fold at P18, PDR3). These data on proliferative capacities mirror earlier reports (32.Reff M. Schneider E.L. Mol. Cell. Biochem. 1981; 36: 169-176Crossref PubMed Scopus (11) Google Scholar, 33.Schneider E.L. J. Invest. Dermatol. 1979; 73: 15-18Abstract Full Text PDF PubMed Scopus (29) Google Scholar), thus validating this use of this cell line for in vitro aging studies. To determine whether the diminutions in absolute and EGF-induced responses that occurred during in vitro aging were mimicked by in vivo aging, dermal fibroblasts from different aged individuals were obtained from the ATCC and NIA repositories. There was variation between individuals, and age of the donor did not strictly predict replicative capacity remaining, as recently reported (34.Cristofalo V.J. Allen R.G. Pignolo R.J. Martin B.G. Beck J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10614-10619Crossref PubMed Scopus (424) Google Scholar). However, cells with lower PDR were less EGF responsive, and this responsiveness decreased further as cells approached senescence (Fig. 1, C and D). A second series of dermal fibroblasts from four similarly aged female donors were qualitatively similar (data for female donors are not shown due to space limitations). The variation in individuals may reflect aging of the fibroblasts, as the cells from the 17-year-old male were already at the near senescent PDR6. As the age of the donor increased, the basal motility and mitogenic levels decreased significantly for fibroblasts from the 83- and 84-year-old donors. EGF responsiveness was retained, although the mitogenic response was diminished (average mitogenic stimulus 4.2-fold in fibroblasts from fetus and baby versusaverage of 2.8-fold in fibroblasts from 83- and 84-year-old donors); the absolute EGF-induced levels were reduced with age. Thus, aged cells lost both cell proliferative and migrative capacity, although these cells retained some EGF-induced cell migration and proliferation responsiveness. This was not unexpected, as near-senescent cells would be selected against during the collection and short term culturing of these donor fibroblasts. The data obtained with the early passage in vivo aged cells suggested that the fibroblasts aged in vivo but had not reached the critical late passage at which we note reduced EGF responsiveness. This would be confirmed by rapid loss of EGF-induced responses upon passaging of these cells. Comparing two fibroblast populations, from the 1-month-old male (CRL-1489) and the 83-year-old male (CRL-7815), the cells from the aged individual senesced earlierin vitro (P7 versus P16; n = 2). The basal cell migrative and proliferative capacities gradually decreased during in vitro aging of the CRL-1489 cells down to levels comparable with the CRL-7815 cells (Fig. 1 C). Late passage cells from either donor lost EGF responsiveness in cell migration (1-month-old (CRL-1489): 1.4–1.2-fold; 83-year-old (CRL-7815): 1.8–1.3-fold) and cell proliferation (CRL-1489: 4.7 to 2.3, CRL-7815: 3.1 to 1.1) compared with earlier passages of the cells (<P12 of CRL-1489, P3 of CRL-7815). Thus, cells aged in vivo presented less reserve in their responsiveness to EGF, a situation that may become limiting during wound healing repopulation. Diminished cellular activities could be due to alterations at any intracellular level from decreased signaling to reduced end-target action. To determine the site of age-related decrease in EGF-induced responses, we assessed activation of downstream signaling pathways and receptor functioning in aged fibroblasts (Fig.2). EGFR kinase activity, including auto-phosphorylation, and Erk1/2-MAPK tyrosyl phosphorylation, as markers of activation (24.Chen P. Xie H. Sekar M.C. Gupta K. Wells A. J. Cell Biol. 1994; 127: 847-857Crossref PubMed Scopus (285) Google Scholar, 35.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4235) Google Scholar), were enhanced by EGF stimulation in early passage Hs68 cells. PLCγ tyrosyl phosphorylation, a surrogate marker of activity (36.Vega Q.C. Cochet C. Filhol O. Chang C.P. Rhee S.G. Gill G.N. Mol. Cell. Biol. 1992; 12: 128-135Crossref PubMed Scopus (79) Google Scholar), was at a high basal level in early passage Hs68 fibroblasts and was only minimally increased by EGF stimulation. In near senescent Hs68 cells (P17) EGF-induced EGFR kinase and auto-phosphorylation are reduced, and there is little if any PLCγ and MAPK tyrosyl phosphorylation. EGF-induced kinase activity and activation of PLCγ and Erk-MAPK are decreased in fibroblasts from the 83-year-old donor compared with those from the fetal or 1-month-old donors (data not shown). However, the signaling is not decreased to the same extent as the near senescent Hs68 cells; this is consistent with the cell response data. The reduced phosphorylation of PLCγ and Erk-MAPK is not due to availability of these effectors, as their levels are essentially unchanged during aging (Fig. 2). We postulated that the reduced EGFR signaling was due to a receptor-level deficit as two divergent pathways were similarly affected, and EGFR levels were shown to be decreased in an earlier report (21.Reenstra W.R. Yaar M. Gilchrest B.A. Exp. Cell Res. 1996; 227: 252-255Crossref PubMed Scopus (42) Google Scholar). Total cellular EGFR levels were assessed using immunoblotting of whole cell lysates (Fig.3 A). EGFR levels in aged cells were down to about half in early population doubling level cells. To determine whether this represented a specific loss of EGFR or whether there was a global decrease in cell surface receptors, the levels of the transferrin receptor and insulin receptor β-subunit were also assessed by immunoblotting and densitometry. These, representing two other classes of surface receptors, were relatively unchanged in aging (Fig. 3 A) when compared with actin, suggesting a specific down-regulation of EGFR levels during cell aging. It was possible that the fewer EGFR were differentially presented, so we assessed the number of binding sites (Fig. 3 B). EGF binding sites decreased during aging down to 40% that of the level of early- and mid-passage cells (Fig. 3 B). The loss of EGFR on the surface mirrored the decrease in total cell EGFR (Fig. 3 A). The decline in EGFR may be due to increased turnover or decreased synthesis. Upon binding, ligand EGFR are rapidly internalized and degraded by a saturable pathway; therefore, we examined internalization of EGF. Internalization was significantly decreased in aged Hs68 and cells from aged individuals (Fig. 4) in agreement with an earlier report (21.Reenstra W.R. Yaar M. Gilchrest B.A. Exp. Cell Res. 1996; 227: 252-255Crossref PubMed Scopus (42) Google Scholar). The internalization of EGFR in near senescent Hs68 (P18, PDR3) was 29% that of young Hs68 (P4, PDR45) and, in the cells from the 83-year-old male, was 44% that of the cells from the 1-month-old male. Thus, increased degradation of EGFR was unlikely to be the cause of decreased levels; rather, the decreased internalization would maintain higher levels of accessible, signaling EGFR. To investigate the effects of cell aging on production of EGFR, mRNA levels were determined (Fig. 5). In near-senescent cells, EGFR mRNA levels were significantly reduced compared with expression of a “housekeeping” gene, GAPDH. The EGFR mRNA expression level of near senescent Hs68 (P18, PDR3) was barely detectable (Fig. 5 A), as was the EGFR mRNA expression level of cells from the 83-year-old male (Fig. 5 B).Figure 5Aging effects on EGFR mRNA expression levels (A
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