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Originally published In Press as doi:10.1074/jbc.M000008200 on April 10, 2000

J. Biol. Chem., Vol. 275, Issue 25, 19343-19351, June 23, 2000
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Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors*

Hidenori Shiraha, Kiran GuptaDagger , Kathryn DrabikDagger , and Alan Wells§

From the Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 and Dagger  Department of Pathology, University of Alabama, Birmingham, Alabama 35294-0007

Received for publication, January 4, 2000, and in revised form, March 16, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, programmed in 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 gamma ) 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, 2). All phases of wound healing are diminished (3, 4). 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). Thus, both fibroblast motility and mitogenesis are critical for wound repair. During aging dermal fibroblasts lose both proliferative and basal migrative capacity (3); 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 (6-10). We propose that understanding the molecular bases underlying this age-associated decline in these capacities will allow for more rational and successful modulation of wound repair.

Fibroblast motility and proliferation are regulated by numerous growth factors (11); among those that are most robust are factors that activate the epidermal growth factor receptor (EGFR)1 (12-16). These factors, transforming growth factor-alpha and heparin-binding EGF-like growth factor in particular (17-19), 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, 21). 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). However, since fibroblasts did not demonstrate a similar correlation between aging and fibroblast growth factor nonresponsiveness (23) 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 gamma  (PLCgamma ) signaling (24, 25). Inhibition of PLCgamma signaling specifically abrogates cell motility but not proliferation (24, 26); 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, 28); the point of divergence of these two cell responses occurs downstream of MAPK kinase in this pathway (28) but remains to be deciphered. Despite the uncertainty of all the signals required for either motility or mitogenesis, these two intracellular effectors, PLCgamma 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 PLCgamma 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- 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). 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).

Cell Proliferation Assay-- EGF-induced proliferation was determined by incorporation of [3H]thymidine by standard procedures (24). 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.

In Vitro Wound Healing Assay-- Basal and EGF-induced migration was assessed by the ability of the cells to move into an acellular area as described previously (15). 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.

Immunoblotting and Immunoprecipitation-- 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-beta -insulin receptor antibody (I16630, Transduction Laboratories, Lexington, KY), anti-transferrin receptor antibody (GR09, Calbiochem), anti-alpha -actin antibody (A-2066, Sigma), anti-phospho-Erk-MAPK (9101, New England Biolabs, Beverly, MA), anti-pan MAPK antibody (9102, New England Biolabs), anti-PLCgamma 1 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 beta -insulin receptor were determined by densitometry (NIH Image) and reported as a ratio to alpha -actin, chosen as a housekeeping gene.

The activation status of PLCgamma 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-PLCgamma 1 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).

EGFR Expression Levels-- The expression level of EGFR was determined by a standard binding assay (30). 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 gamma -counter (Beckman Instruments). The number of binding sites was calculated by Scatchard analysis using linear regression.

mRNA Analyses-- 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). A probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was prepared from the human GAPDH cDNA (ATCC). The probes were labeled with alpha [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.

Nuclear Run-on Assay-- 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.

Cyclic AMP (cAMP) Assay-- Cells were plated in 10-cm culture plate and grown to confluence in DME with 10% FBS. Cells were treated with interferon-gamma -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 N HCl) 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.

Expression of Exogenously encoded EGFR-- 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). 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 107 cells 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.

Determination of EGFR Internalization-- 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 nM 125I-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 bound 125I-EGF was obtained by collecting two washes of the cells with acid strip buffer (50 mM glycine, 100 mM NaCl, 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 gamma  counter (Beckman). The endocytic rate constants were calculated by the time course of loss of surface-bound EGF and accumulation of internalized EGF (31).

Bromodeoxyuridine (BrdUrd) Staining-- 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)

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Aged Hs68 Present Reduced Basal and EGF-induced Migrative and Cell Proliferative Capacities-- 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 (<PDR10) (Fig. 1A). The average basal cell motility decreases steadily from 360 µm/day in early passage down to 290 µm/day in near senescent fibroblasts, although only by P18 (PDR3) was there a statistical difference in motility. EGF-induce motility was relatively constant until about P13 (PDR18) at >470 µm/day (average 1.7-fold induction); thereafter it fell precipitously to 300 µm/day by P18 (PDR3) (1.1-fold induction).


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  		Fig. 1.   Effect of in vitro (A and B) and in vivo (C and D) aging on fibroblast motility (A and C) and proliferation (B and D). A and B, Hs68 were passaged and tested at different passages with PDR back-calculated. Cell migration assays and cell proliferation assays were performed in the absence (open circle  and open bars) and presence ( and black bars) of EGF (1 nM) as described under "Experimental Procedures." The data are the mean ± S.E. of more than three independent studies, each performed in triplicate. Statistical analysis was performed by Student's t test as compared with P5 (PDR42) of Hs68: *, p < 0.05, **, p < 0.01 C, fibroblasts from fetus male (triangle , basal; black-triangle, with EGF), 1-month-old male (CRL-1489) (, basal; black-square, with EGF), 17-year-old male (CRL-7315) (diamond , basal; black-diamond , with EGF), and 83-year-old male (CRL-7815) (open circle , basal; , with EGF) were assessed by cell migration assay. Fibroblasts from 1-month-old male (CRL-1489) and 83-year-old male (CRL-7815) were passaged and assessed at indicated passages. Basal and EGF-induced cell motility were measured as described under "Experimental Procedures." The data are the mean of more than three independent studies, each performed in triplicate. Statistical analysis was preformed by Student's t test as compared with P8 (PDR24) of CRL-1489 or P3 (PDR12) of CRL-7815: *, p < 0.05, **, p < 0.01 D, fibroblasts from different aged donor were obtained. Basal (clear bars) and EGF-induced (black bars) thymidine incorporation were measured as described under "Experimental Procedures." The data are the mean ± S.E. of more than three independent studies performed in triplicate. Statistical analysis was performed by Student's t test as compared with fetal cells: *, p < 0.05, **, p < 0.01

Basal thymidine incorporation remained low but steady during early- and mid-passage (average 8300 cpm) until late passage (P18, PDR3 presented 850 cpm) (Fig. 1B). 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, 33), thus validating this use of this cell line for in vitro aging studies.

Cells from Aged Individuals Present Lower Basal Capacities and Lose EGF Responsiveness Sooner-- 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). 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 versus average 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 earlier in 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. 1C). 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.

EGFR Signaling and Expression Levels Are Decreased in Aged Fibroblasts-- 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, 35), were enhanced by EGF stimulation in early passage Hs68 cells. PLCgamma tyrosyl phosphorylation, a surrogate marker of activity (36), 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 PLCgamma and MAPK tyrosyl phosphorylation. EGF-induced kinase activity and activation of PLCgamma 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 PLCgamma and Erk-MAPK is not due to availability of these effectors, as their levels are essentially unchanged during aging (Fig. 2).


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  		Fig. 2.   Tyrosyl phosphorylation of intracellular targets of EGF receptor signaling in Hs68. Cells were treated with or without 1 nM EGF for 5 min. Cells were lysed, and equal volumes of cell lysates were size-fractionated by 7.5% SDS-PAGE and immunoblotted with antibodies specific for phosphotyrosine (PY-20, Transduction Laboratories), PLCgamma (#05-163, Upstate Biotechnology Inc.), phospho-Erk-MAPK (#9101, New England Biolabs), or pan-Erk-MAPK (#9102, New England Biolabs). In the phospho-PLCgamma analysis, cells were treated in the absence or presence of 1 nM EGF for 5 min. Cell lysates were immunoprecipitated with the anti-PLCgamma antibody, size-fractionated by SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody. Shown are representative blots of at least two repeats at all data points.

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). Total cellular EGFR levels were assessed using immunoblotting of whole cell lysates (Fig. 3A). 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 beta -subunit were also assessed by immunoblotting and densitometry. These, representing two other classes of surface receptors, were relatively unchanged in aging (Fig. 3A) 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. 3B). EGF binding sites decreased during aging down to 40% that of the level of early- and mid-passage cells (Fig. 3B). The loss of EGFR on the surface mirrored the decrease in total cell EGFR (Fig. 3A).


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  		Fig. 3.   EGFR expression levels in different aged cells. A, cells were grown to confluence and lysed. Cell lysates were size-fractionated by 7.5% SDS-PAGE and immunoblotted with anti-EGFR (#05-104, Upstate Biotechnology Inc.), anti-transferrin receptor (GR09, Calbiochem), anti-insulin receptor beta -subunit (I16630, Transduction Laboratories), or anti-alpha -actin antibodies (A-2066, Sigma). EGFR and other receptor expression levels were enumerated by densitometry and calculated as a ratio of alpha -actin. Representative blots are shown of at least two repeat experiments. The receptor expression levels were determined as the ratio to alpha -actin level by densitometry analysis. The data are the mean ± S.E. of more than two independent studies. Statistical analysis was performed by Student's t test as compared with P5 of Hs68: *, p < 0.05, **, p < 0.01 B, EGF binding sites were enumerated by standard Scatchard analyses as described under "Experimental Procedures." The numbers of binding sites were calculated by Scatchard plot using linear regression. Shown are representative values; all values were determined twice except for CRL-1489. The values for Hs68 are the means of more than two independent studies. open circle , Hs68; triangle , cells from fetus male (CRL-1475); , cells from 1-month-old male (CRL-1489); ×, cells from 17-year-old male (CRL-7315); diamond , cells from 83-year-old male (CRL-7815).

EGFR Synthesis Is Decreased in Aged Cells-- 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). 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. 5A), as was the EGFR mRNA expression level of cells from the 83-year-old male (Fig. 5B).


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  		Fig. 4.   Ligand-induced internalization of EGFR in in vitro aged Hs68 fibroblasts (A) and fibroblasts from male donors (B). Internalized and surface-bound EGF were determined using 125I-EGF as described under "Experimental Procedures." The endocytic rate constants were calculated by the time course of loss of surface-bound EGF and accumulation of internalized EGF. The data are the mean ± S.E. of at least two experiments at each point except for CRL-7815. Statistical analysis was performed by Student's t test as compared with early passage of cells: **, p < 0.01.


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  		Fig. 5.   Aging effects on EGFR mRNA expression levels (A and B) and stability (C). A, B. The expression levels of EGFR were determined by Northern blot analyses. Shown are representative blots of at least two blots for each situation. C, RNAs were collected before and after 2.5 h of treatment with actinomycin D (5 µM). The amount of mRNA for EGFR and GAPDH were determined by Northern blot analyses and phosphoimaging analyzer (Bio-Rad). The data area shown is the ratio to P6 of Hs68 without actinomycin D, shown in all the blots is the ~5.8 kilobase mRNA species that represented the overwhelmingly predominant band in all experiments.

We also assessed the effect of aging on EGFR mRNA stability. The mRNA transcription inhibitor actinomycin D treatment caused a 19% and 15% decrease of EGFR mRNA on P6 (PDR39) and P18 (PDR3), respectively (Fig. 5C). As the near senescent cells do not display greater instability than the early passage cells, cell aging does not seems to affect on EGFR mRNA stability. Transcriptional activity of EGFR mRNA was assessed by nuclear run-on assay. EGFR mRNA transcriptional level was decreased to ~50% in aged Hs68 (Fig. 6). The correlation of reduced mRNA and protein levels suggest that the age-related loss in EGFR is effected at the RNA transcription level.


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  		Fig. 6.   Aging effect on gene transcription. The transcriptional levels of EGFR and the other genes were measured by nuclear run-on assay. Intact nuclei were isolated by Nonidet P-40 buffer. 32P-labeled RNAs were extracted and hybridized with indicated dot-blotted DNA. The gene transcriptional level was determined by densitometry. The data are the mean ± S.E. of three independent studies. The data were shown as the ratio to GAPDH mRNA transcription. Statistical analysis was performed by Student's t test as compared with P5 of Hs68: *, p < 0.05. The picture shown is a composite picture that was generated by overlaying all three experiments after adjusting the GAPDH density; in each assay EGFR transcriptional rates were significantly lower in the low PDR cells. IR, insulin receptor beta -subunit; TR, transferrin receptor.

cAMP Signaling Is Relatively Maintained in Aging Fibroblasts-- To confirm that some of the signaling pathways from the cell surface are maintained in aging, we determined cAMP generation in response to a pharmacological and a physiological agent. Forskolin induced a robust increase in intracellular cAMP in both young (2.5-fold in PDR42) and near senescent Hs68 cells (2.3-fold in PDR3) (Fig. 7). The anti-motility ELR-negative CXC chemokine interferon-gamma -inducible protein-10 (37) also elicited a strong response in both young and near senescent Hs68 cells (2.0-fold and 1.8-fold, respectively). Thus, there does not appear to be a global decrease in all signaling pathways from the cell surface.


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  		Fig. 7.   Total cellular cAMP induction. P5 (PDR42) and P18 (PDR3) of Hs68 were grown to confluent in 10-cm culture dish. After the 4-h treatment of interferon-gamma -inducible protein-10 (IP-10; 50 ng/ml) or a 30 min treatment of forskolin (25 µM), samples were extracted with extraction buffer (50% ethanol, 0.1 N HCl). The extracts were lyophilized and re-suspended in water. The samples were analyzed using cAMP assay kit (Amersham Pharmacia Biotech). The data are shown as the ratio to mock-treated (nTx; control) cells. The data are the mean ± S.E. of three independent studies, each performed in duplicate. Statistical analysis was performed by Student's t test as compared with P5 of Hs68. No significant decrease was found in P18 of Hs68.

EGFR Re-expression Restores the EGF-induced Cell Migrative and Proliferative Capacities-- We hypothesized that decreased EGFR levels are cause for loss of EGF-induced responses in near senescent fibroblasts. The foregoing data could simply reflect a linked but not causal phenomenon of growth factor receptors decreasing during cell aging. To test this, near senescent Hs68 cells (P18, PDR3) were electroporated with cDNA encoding either EGFR or GFP (as control). About 44% of the GFP electroporated cells expressed GFP after 48 h. Despite the only partial transfection, the EGFR expression level of EGFR plasmid-targeted cells was about twice that of GFP control cells (Fig. 8), suggesting that the transfected cells expressed EGFR near the level of non-aged fibroblasts. The EGFR plasmid-targeted cells presented more EGF binding sites, as determined by Scatchard analysis (6.3 × 104/cell compared with 3.4 × 104/cell), in parallel with total EGFR levels as determined by immunoblotting and densitometry analysis (1.8-fold; Fig. 8D). These cells recovered, at least in part, the EGF-induced motility and proliferation responses compared with GFP cells (motility response increased from 1.1-fold to 2.0-fold and mitogenic response from 1.7-fold to 2.4-fold; n = 3). The lack of a complete recovery may be due to the only partial electroporation combined with the transient nature of EGFR expression.


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  		Fig. 8.   Expression of exogenously encoded EGFR in near senescent Hs68 fibroblasts (C and D) and effects on cell motility (A) and mitogenesis (B). Eukaryotic expression plasmids for EGFR or GFP (control) were introduced into P18 (PDR3) of Hs68 using electroporation technology (Gene Pulser, Bio-Rad). The cells were incubated for 48 h in DME containing 0.1% dialyzed FBS before analyses as described under "Experimental Procedures." A, cell migration assay; B, thymidine incorporation; C, immunoblot analysis with anti-phosphotyrosine antibody phosphotyrosine (PY-20, Transduction Laboratories); D, immunoblot analysis and densitometry using anti-EGFR (#05-104, Upstate Biotechnology Inc.) or anti-alpha -actin (A-2066, Sigma) antibodies. The data in graphs (A) and (B) are the mean ± S.E. of three independent electroporations, with each experiment performed in triplicate. The data in (C) and (D) are representative of the electroporation experiments. Statistical analysis was performed by Student's t test as compared with control cells: *, p < 0.05, **, p < 0.01.

This recovery of responsiveness was also observed in near senescent (P6, PDR3) fibroblasts from the 83-year-old male donor (CRL-7815). 40% of the GFP cells expressed after 48 h. These cells demonstrated recovery of EGF-induced responsiveness in both cell motility and proliferation (motility response increased from 1.1-fold to 1.4-fold; mitogenic response increased from 1.3-fold to 1.5-fold; n = 3). To better determine that the cells expressing higher, "young" levels of EGFR contributed to the EGF responsiveness, we attempted to evaluate single cells. Near senescent cells were co-electroporated with EGFR and GFP plasmid (DNA molar ratio 7.5:1); GFP-positive cells were presumed to express the exogenous EGFR. GFP-expressing cells were over-represented in the cells that migrated from the denuded front (68% of migrating cells versus 40% in the population) and appeared to migrate further. BrdUrd staining was performed to assess proliferative activity. Cells were electroporated with or without EGFR (20 µg) and GFP (20 µg). GFP-positive cells were marked by BrdUrd incorporation at 1.5 times the fraction as GFP-negative cells (1.46- and 1.49-fold, n = 2); this ratio is similar to the increase in thymidine incorporation noted in mass cultures. The results of the single cell analyses mirror the population studies and strongly suggest that the restoration of EGF responsiveness was due to the EGFR-re-expressing cells.

Increased EGFR Signaling Capacity Does Not Enhance the Cell Migrative Activity in Young Cells-- Our finding that re-expression of EGFR in near senescence cells restored EGF responsiveness could have been due to a simple dose-response effect if responsiveness was linearly related to the EGFR level. Thus, we assessed the effect of increased EGFR signaling capacity in young cells. Early passage of Hs68 (P5, PDR42) was electroporated with EGFR or GFP (as control). About 45% of GFP eletroporated cells expressed GFP after 48 h. Although EGFR electroporated cells expressed more EGFR, these cells did not increase EGF-induced cell migrative capacity compared with GFP cells (Fig. 9, A and B). Furthermore, electroporation of excessive EGFR cDNA led to ligand-induced cytotoxicity, as described in the literature for supraphysiological levels of EGFR (38). A second method for increasing EGFR signaling would be to use supersaturating concentrations of ligand (10 nM) in lieu of our usual dose that approximates Kd (1 nM). Increasing the EGF concentration 10-fold had no effect on the response of near senescent cells. Early passage cells did not demonstrate increased responsiveness to high levels of ligand (Fig. 9C). Thus, merely increasing EGFR signaling capacity in these cells does not lead to increased responsiveness, suggesting a qualitatively distinct mechanism for loss of responsiveness.


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  		Fig. 9.   Effect of increased EGFR signaling capacity on early passage of Hs68 fibroblasts. A and B. EGFR (10 µg/107 cells) or GFP (20 µg/107 cells as control) plasmid were introduced into P5 (PDR42) Hs68 cells. The cells were incubated for 48 h in DME containing 0.1% dialyzed FBS before analyses as described under "Experimental Procedures." A, cell migration assay; B, immunoblot analysis with anti-phosphotyrosine antibody phosphotyrosine (PY-20, Transduction Laboratories), anti-EGFR (Upstate Biotechnology Inc.) or anti-alpha -actin (Sigma) antibodies. The data are the mean ± S.E. of two independent electroporations, with each experiment performed in triplicate. The blots shown are representative of the electroporation experiments. C, in vitro wound healing assays were performed with P5 of Hs68 in the absence and presence of EGF (1 or 10 nM). The data are the mean ± S.E. of more than two independent studies, each performed in triplicate. There were no statistical differences in motility responses between the EGF treatments in A and C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented herein suggest a model of dermal fibroblast aging in which both basal and EGF-stimulated cell activities decline with decreasing PDR. The loss of EGF responsiveness in near senescent cells is linked to decreased level of EGFR production. The decrease in basal cell activities is likely due to a second, possibly independent cell change that does not involve EGFR signaling. It remains to be determined whether these two events have a common causal basis at the gene regulation level.

During in vitro aging, the decline in EGFR was reflected by loss of EGF responsiveness for both migration and proliferation responses. This occurred most acutely in near senescent fibroblasts. The loss of two distinct and competing cell responses (14, 24), along with decreased activation of two divergent intracellular signaling pathways despite unchanged levels of protein, suggested that the reduced EGFR was cause for the response deficit.

Age-related diminution in non-stimulated dermal fibroblast motility has been attributed to global cell changes (23, 39, 40) and would thus be independent from the loss of EGF responsiveness noted in near senescent cells. This fits with our findings on early passage fibroblasts from aged donors; basal motility decreases, whereas EGF responsiveness is retained despite declining levels of EGFR. On the other hand, autocrine growth factor signaling has been proposed as stimulating dermal fibroblast (41), although this has yet to be documented for EGFR signaling (42), raising the possibility that the EGFR decreases underlies both phenomena. At present, we cannot eliminate the possibility that some level of autocrine signaling through EGFR occurs in these fibroblasts and that the decreasing motility and mitogenesis noted in the absence of exogenously added EGF reflects a parallel decrement in this EGFR-mediated signaling (43). In fact, the unstimulated levels of PLCgamma and Erk-MAPK activation are high compared with mouse immortalized fibroblasts (24) and do decline in parallel with age-related reduction in EGFR. Still, we do not favor this latter scenario, since the early passage dermal fibroblasts from different aged donors all present EGF-induced motility despite significantly reduced EGFR levels on the cells from the aged donors.

We found that EGFR levels preferentially declined during cellular aging regardless of whether the cellular aging occurred in vivo and in vitro. This was noted as loss in ligand binding sites per cell and as total cellular EGFR protein. Thus, this loss of responsiveness does not merely reflect altered trafficking, delayed glycoprotein maturation and transit, or increased endocytosis and retention. We investigated the underlying mechanism for decreased EGFR. Paradoxically, the data show that receptor internalization is diminished with aging, as previously reported (21), as if to spare the few receptors from down-regulation since signaling from the surface is sufficient to elicit both the biochemical and physiological events leading to the cell responses of mitogenesis and motility (44, 45). On the other hand, EGFR mRNA levels were diminished in parallel with reduced surface and total EGFR. That this is at least semi-specific for EGFR is demonstrated by comparing EGFR protein levels to the cytoskeletal protein actin and two cell surface receptors, the receptors for insulin and transferrin. These latter represent two biologically and biochemically distinct receptor classes. Although an age-related transcriptional control element remains to be identified, the results of our nuclear run-on experiment suggest that this preferential loss of EGFR seems to be caused by the decrement of mRNA transcriptional activity.

In addition to EGFR, other growth factor receptors are also decreased in aging cells; these include receptors for fibroblast growth factor and platelet-derived growth factor (22, 23, 43, 46). It is possible that the diminished levels of these might be controlled by similar mechanism as EGFR. Our data show EGFR expression levels parallel PDR number, not necessarily donor age, as might be expected from earlier reports (34, 47). In addition, the PLCgamma and Erk1/2-MAPKs, representing two signaling pathways that diverge from the receptor (24), also were relatively unchanged. These observations do not eliminate the possibility of other motility-signaling molecules also being specifically down-regulated, especially in other cell types, as might be the situation for aged hepatocytes that appear to be deficient in EGFR coupling to Shc (48). Thus, our findings do highlight EGFR expression as a marker of age-related alterations in the fibroblast cell proteome.

These data show that decreased functioning in aged cells is due not to a global deficit but to specific deficits. Signaling from some cell surface receptors is decreased in aging (46), and this decrease is likely controlled separately from global cell dysfunction. To support this, we found that insulin receptor levels were relatively unchanged in near senescent Hs68 cells and, in parallel, the insulin responsiveness in cell migrative and cell proliferative capacities were relatively unchanged. However, insulin had very little effect on cell migrative and cell proliferative capacities compared with EGF in Hs68 fibroblast (data not shown). To confirm that some signaling pathways from cell surface receptors were maintained in the face of near senescence, we assessed the effect of forskolin and interferon-gamma -inducible protein-10 on the generation of intra-cellular cAMP (37). The near senescent cells responded similarly to the young Hs68 cells. Thus, at least some signaling pathways are intact in aged cells.

Decreased levels of EGFR appeared to be the underlying mechanism for loss of responsiveness to EGF in aged cells. As this was reflected as diminished transcription and not increased consumption, we could correct this deficit by introducing EGFR cDNA under an autonomous promoter. Aged cells were successfully electroporated to express EGFR levels similar to "young" cells; these cells regained their EGFR responsiveness. These experiments demonstrate for the first time that specific cellular response deficits to factors in near senescent cells can be corrected by restoring a single gene product. Although not advocating such an approach, these data support the use of EGFR gene transfer to nonhealing wounds as a more rational therapy than application of growth factors, since the rate-limiting step is receptor signaling and not factor availability (although specific growth factors are reduced in wounds in aged patients (43)). Furthermore, we do not know whether introduction of EGFR into senescent cells will restore any responses, as we were unable to stably introduce EGFR cDNA either under constitutive or inducible transcriptional controls. In fact, senescence is likely independently regulated from EGFR levels and responsiveness. We and others (20, 21, 49, 50) also noted an age-related decrease in basal cell mitogenesis and motility. Whether this was related to the loss of EGFR signaling could be tested in immortalized human fibroblasts that had been rescued from senescence by stable expression of telomerase (51). We determined that these cells had not regained the EGF responsiveness associated with non-senescent dermal fibroblasts by comparing these cells to late passage controls. However, this was confounded by finding that average basal cell motility and thymidine incorporation was higher in two different human telomerase-positive cell populations (510 µm/day compared with 310 µm/day and 9200 cpm compared with 2900 cpm). The EGF responsiveness of the immortalized cells was, if anything, reduced when compared with the two aged control fibroblast populations (1.3-fold compared with 2.6-fold). This may reflect the fact that the expression levels of EGFR in human telomerase-positive cells were not higher, and possibly lower, than in the human telomerase negative control cells. These data point to the multiple, possibly independently regulated alterations during aging that lead to diminished cell activities. Thus, our model remains one of a global cell alteration reducing cell responsiveness gradually with aging, with an acute loss of EGF responsiveness superimposed as the cells approach senescence.

EGFR re-expressed cells recover robust EGF responsiveness, although the EGF-induced cell mitogenesis is still lower than that of early passage cells or cells from neonatal donors. The partial recovery of cell responses may be due to incomplete electroporation efficiency combined with the transient expression or to age-related decrements in general cell functioning such as protein synthesis (52-55), which is involved in cell motility (15, 56) and mitogenesis (35). The ramification of these findings for wound healing are obvious. In aged mammals there is a deficit in all phases of dermal wound healing (3). During the reparative phase, for which fibroblast repopulation is critical (57), the age-related decrease in motility and proliferation would only briefly be compensated by enhanced activities induced by EGFR ligands in the wound field. As these cells would then rapidly approach senescence upon mitogenesis, this would be lost. As aged fibroblasts have been reported to produce less matrix (58), the consequence would be reduced dermal tissue and a weaker scar, which is what is seen in aged individuals. To overcome this deficit, many extracellular factors have been applied to wounds with only limited success (6-10, 59), which would be explained by an intrinsic cell defect. The implications are that interventions to improve healing in aged skin need to be targeted toward improving cell responsiveness by enhancing cell signaling and augmenting the cell responses.

    ACKNOWLEDGEMENTS

We thank Philip Chang, Jareer Kassis, and Hyung Kim for valuable suggestions and Drs. W. E. Wright (University of Texas Southwestern Medical Center) and K. R. Kaster (Geron Corp.) for generously providing the human telomerase-expressing cells. Figure preparation was assisted by Birmingham Veterans Affairs Medical Center.

    FOOTNOTES

* This work was supported by National Institutes of Health (NIGMS) Grant GM54739.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Pathology, University of Pittsburgh, S713 Scaife, Terrace St., Pittsburgh, PA 15261. Tel.: 412-624-0973; Fax: 412-647-8567; E-mail: wellsa@msx.upmc.edu.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M000008200

    ABBREVIATIONS

The abbreviations used are: EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescence protein; MAPK, mitogen-activated protein kinase; PDR, population doublings remaining; PLCgamma , phospholipase C-gamma ; Erk, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; BrdUrd, bromodeoxyuridine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Lober, C. W., and Fenske, N. A. (1991) South. Med. J. 84, 1444-1446
2. Nicolle, L. E., Huchcroft, S. A., and Cruse, P. J. (1992) J. Clin. Epidemiol. 45, 357-364
3. Ashcroft, G. S., Horan, M. A., and Ferguson, M. W. (1995) J. Anat. 187, 1-26
4. Gerstein, A. D., Phillips, T. J., Rogers, G. S., and Gilchrest, B. A. (1993) Dermatol. Clin. 11, 749-757
5. Hay, E. D. (1993) Curr. Opin. Cell Biol. 5, 1029-1035
6. Beck, L. S., DeGuzman, L., Lee, W. P., Xu, Y., Siegel, M. W., and Amento, E. P. (1993) J. Clin. Invest. 92, 2841-2849
7. Bennett, N. T., and Schultz, G. S. (1993) Am. J. Surg. 165, 728-737
8. Brown, G. L., Nanney, L. B., Griffen, J., Cramer, A. B., Yancey, J. M., Curtsinger, L. J., Holtzin, L., Schultz, G. S., Jurkiewicz, M. J., and Lynch, J. B. (1989) N. Engl. J. Med. 321, 76-79
9. Hennessey, P. J., Nirgiotis, J. G., Shinn, M. N., and Andrassy, R. J. (1991) J. Pediatr. Surg. 26, 362-365
10. Schultz, G., Khaw, P. T., Oxford, K., MaCauley, S., Van Setten, G., and Chegini, N. (1994) Eye 8, 184-187
11. Lawrence, W. T., and Diegelmann, R. F. (1994) Clin. Dermatol. 12, 157-169
12. Barrandon, Y., and Green, H. (1987) Cell 50, 1131-1137
13. Blay, J., and Brown, K. D. (1985) J. Cell. Physiol. 124, 107-112
14. Carpenter, G., and Cohen, S. (1978) Natl. Cancer Inst. Monogr. 48, 149-156
15. Chen, P., Gupta, K., and Wells, A. (1994) J. Cell Biol. 124, 547-555
16. Gospodarowicz, D., and Mescher, A. L. (1977) J. Cell. Physiol. 93, 117-127
17. Blotnick, S., Peoples, G. E., Freeman, M. R., Eberlein, T. J., and Klagsbrun, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2890-2894
18. Kiritsy, C. P., Lynch, A. B., and Lynch, S. E. (1993) Crit. Rev. Oral Biol. Med. 4, 729-760
19. Steenfos, H. H. (1994) Scand. J. Plast. Reconstr. Surg. Hand Surg. 28, 95-105
20. Liu, Y., Guyton, K. Z., Gorospe, M., Xu, Q., Kokkonen, G. C., Mock, Y. D., Roth, G. S., and Holbrook, N. J. (1996) J. Biol. Chem. 271, 3604-3607
21. Reenstra, W. R., Yaar, M., and Gilchrest, B. A. (1996) Exp. Cell Res. 227, 252-255
22. Garfinkel, S., Hu, X., Prudovsky, I. A., McMahon, G. A., Kapnik, E. M., McDowell, S. D., and Maciag, T. (1996) J. Cell Biol. 134, 783-791
23. Garfinkel, S., Wessendorf, J. H., Hu, X., and Maciag, T. (1996) Biochim. Biophys. Acta 1314, 109-119
24. Chen, P., Xie, H., Sekar, M. C., Gupta, K., and Wells, A. (1994) J. Cell Biol. 127, 847-857
25. Ji, Q. S., Ermini, S., Baulida, J., Sun, F. L., and Carpenter, G. (1998) Mol. Biol. Cell 9, 749-757
26. Chen, P., Xie, H., and Wells, A. (1996) Mol. Biol. Cell 7, 871-881
27. Pages, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, J., and Pouyssegur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8319-8323
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., and Wells, A. (1998) J. Cell Sci. 111, 615-624
29. Cristofalo, V. J., Charpentier, R., and Phillips, P. D. (1998) in Cell Biology: A Laboratory Handbook (Celis, J. E., ed) , pp. 313-318, Academic Press, Inc., San Diego
30. Wiley, H. S., Walsh, B. J., and Lund, K. A. (1989) J. Biol. Chem. 264, 18912-18920
31. Wiley, H. S., and Cunningham, D. D. (1981) Cell 25, 433-440
32. Reff, M., and Schneider, E. L. (1981) Mol. Cell. Biochem. 36, 169-176
33. Schneider, E. L. (1979) J. Invest. Dermatol. 73, 15-18
34. Cristofalo, V. J., Allen, R. G., Pignolo, R. J., Martin, B. G., and Beck, J. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10614-10619
35. Marshall, C. J. (1995) Cell 80, 179-185
36. Vega, Q. C., Cochet, C., Filhol, O., Chang, C. P., Rhee, S. G., and Gill, G. N. (1992) Mol. Cell. Biol. 12, 128-135
37. Shiraha, H., Glading, A., Gupta, K., and Wells, A. (1999) J. Cell Biol. 146, 243-254
38. Kawamoto, T., Mendelsohn, J., Le, A., Sato, G. H., Lazar, C. S., and Gill, G. N. (1984) J. Biol. Chem. 259, 7761-7766
39. Phillips, G. D., and Stone, A. M. (1994) Mech. Ageing Dev. 73, 189-196
40. Pienta, K. J., and Coffey, D. S. (1990) Mech. Ageing Dev. 56, 99-105
41. Kondo, H., and Yonezawa, Y. (1992) Mech. Ageing Dev. 63, 223-233
42. Kondo, H., Yonezawa, Y., and Ito, H. (1996) Mech. Ageing Dev. 87, 141-153
43. Ashcroft, G. S., Horan, M. A., and Ferguson, M. W. (1997) J. Anat. 190, 351-365
44. Wells, A., Welsh, J. B., Lazar, C. S., Wiley, H. S., Gill, G. N., and Rosenfeld, M. G. (1990) Science 247, 962-964
45. Haugh, J. M., Schooler, K., Wells, A., Wiley, H. S., and Lauffenburger, D. A. (1999) J. Biol. Chem. 274, 8958-8965
46. Aoyagi, M., Fukai, N., Ogami, K., Yamamoto, M., and Yamamoto, K. (1995) J. Cell. Physiol. 164, 376-384
47. Hollenberg, M. D., and Schneider, E. L. (1979) Mech. Ageing Dev. 11, 37-43
48. Palmer, H. J., Tuzon, C. T., and Paulson, K. E. (1999) J. Biol. Chem. 274, 11424-11430
49. Carlin, C. R., Phillips, P. D., Knowles, B. B., and Cristofalo, V. J. (1983) Nature 306, 617-620
50. Ishigami, A., Reed, T. D., and Roth, G. S. (1993) Biochem. Biophys. Res. Commun. 196, 181-186
51. Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S., and Wright, W. E. (1998) Science 279, 349-352
52. Burns, A. L., and Kaulenas, M. S. (1979) Mech. Ageing Dev. 11, 153-169
53. Meerson, F. Z., Javich, M. P., and Lerman, M. I. (1978) J. Mol. Cell. Cardiol. 10, 145-159
54. Newman, R. A., and Langner, R. O. (1975) Connect. Tissue Res. 3, 231-236
55. Viskup, R. W., Baker, M., Holbrook, J. P., and Penniall, R. (1979) Exp. Aging Res. 5, 487-496
56. Goldschmidt-Clermont, P. J., Kim, J. W., Machesky, LM, Rhee, S. G., and Pollard, T. D. (1991) Science 251, 1231-1233
57. Clark, R. A. F. (1996) The Molecular and Cellular Biology of Wound Repair , pp. 22-34, Plenum Publishing Corp., New York
58. Colige, A., Nusgens, B., and Lapiere, C. M. (1990) J. Cell. Physiol. 145, 450-457
59. Benn, S. I., Whitsitt, J. S., Broadley, K. N., Nanney, L. B., Perkins, D., He, L., Patel, M., Morgan, J. R., Swaim, W. F., and Davidson, J. M. (1996) J. Clin. Invest. 98, 2894-2902

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