to emphasize that the serum-induced transcription of c-fos is specifically repressed ... cells into adipocytes represses growth factor responsiveness by limiting the ...
RESEARCH ARTICLE
1011
Senescence represses the nuclear localization of the serum response factor and differentiation regulates its nuclear localization with lineage specificity Wei Ding, Sizhi Gao and Robert E. Scott* Department of Pathology, University of Tennessee Health Science Center, 3 N. Dunlap Street, Memphis, TN 38163, USA *Author for correspondence
Accepted 30 November 2000 Journal of Cell Science 114, 1011-1018 © The Company of Biologists Ltd
SUMMARY The differentiation of cultured 3T3T mesenchymal stem cells into adipocytes represses growth factor responsiveness by limiting the nuclear localization of the serum response factor (SRF) that binds to and activates the promoters of growth control genes that contain the serum response elements (SRE), such as junB and c-fos. The regulation of SRF nuclear localization by adipocyte differentiation is specific, because we show that adipocyte differentiation does not repress the nuclear localization of six other transacting factors. To determine if repression of growth factor responsiveness that occurs during senescence also represses the nuclear localization of SRF, we studied normal human WI-38 fibroblasts at low versus high population doublings. The results show that SRF localizes to the nucleus of proliferative cells whereas in senescent cells SRF can not be detected in the nucleus. This result is apparent in both immunofluorescence assays and in
INTRODUCTION The relationship between cell proliferation and differentiation has been extensively studied. The induction of differentiation typically is associated with the progressive loss of proliferative potential that leads to terminal differentiation (Potten and Lajtha, 1982; Till, 1982). The signaling pathways by which differentiation is induced and the factors that induce differentiation however can show lineage specificity. For example, in murine mesenchymal cells, serum induces proliferation, whereas plasma induces differentiation (Hoerl and Scott, 1989). In contrast, in human epithelial keratinocytes, serum induces differentiation rather than proliferation (Wille et al., 1984). Cellular senescence resembles terminal differentiation in that both processes cause cells to irreversibly lose the ability to proliferate in response to mitogenic agents (Wier and Scott, 1986). Replicative senescence of normal human diploid cells occurs after a limited number of cell divisions (Hayflick and Moorhead, 1961; Hayflick, 1965; Goldstein, 1990). The number of cell divisions after which senescence occurs has a direct inverse correlation with the age of the donor. Cells from patients afflicted with premature aging syndromes also show a reduced in vitro lifespan (Stanulis-Praeger, 1987). Therefore, senescence in culture mimics aspects of the aging process in
western blot analysis. We next evaluated the cellular distribution of SRF in selected human tissues to determine whether the loss of proliferative potential in vivo could have a different effect on SRF nuclear localization. We found that in cells of the small bowel mucosa, differentiation modulates SRF nuclear localization in an opposite manner. Minimal SRF expression and nuclear localization is evident in undifferentiated cells at the base of crypts whereas increased SRF expression and nuclear localization is evident in differentiated cells at the surface tip of the villus. These results together establish that regulation of SRF expression and nuclear localization is important in senescence and differentiation in a lineage specific manner. Key words: SRF, Differentiation, Senescence, Nuclear localization, Murine 3T3T cell, Human fibroblast, Human small bowel mucosal cell
vivo. Of the many theories proposed to explain senescence, one that is believed to be most plausible proposes that telomere loss acts like a molecular clock that can keep track of cell division numbers (Campisi, 1997; Sedivy, 1998). Since telomeres shorten with each cell cycle in normal somatic cells, a critical shortening in the length of telomeric DNA is thought to signal irreversible growth arrest. Once cells enter the senescent state, the expression of several classes of genes changes, including cell cycle regulatory cyclins, CDKs and CDK-inhibitors (Wong and Riabowol, 1996), the tumor suppressor genes Rb-1 and p53 (Jansen-Durr, 1998; Vaziri and Benchimol, 1999), and the serum-inducible gene c-fos. With regard to the current studies, it is important to emphasize that the serum-induced transcription of c-fos is specifically repressed by senescence (Seshadri and Campisi, 1990). Nevertheless, the SV40 large T antigen can induce both DNA synthesis and c-fos expression in senescent cells (Campisi, 1992). Overexpression of c-fos in senescent cells can also induce limited DNA synthesis (Phillips et al., 1992), while microinjection of anti-sense c-fos oligonucleotide into senescent cells can block the T antigen-induced DNA synthesis (Campisi, 1992). These results indicate that c-fos plays an important role in senescence. The repression of c-fos transcription also occurs during the adipocyte differentiation of 3T3T mesenchymal stem cells (Wang and Scott, 1994). More
1012
JOURNAL OF CELL SCIENCE 114 (5)
specifically, we reported that when 3T3T adiopcytes first develop a differentiated phenotype, three events happen. Serum and growth factor responsiveness is repressed, AP-1 DNA binding activity is repressed and the transcription of c-fos and junB is repressed. The c-fos promoter has been studied extensively. Among several regulatory sequences on the c-fos promoter, the serum response element (SRE) appears to be most important in mediating the transcriptional induction of c-fos triggered by various growth factors, serum and stress stimuli (Treisman, 1990). The c-fos SRE is a 20 base pairs dyad symmetry element which is specifically recognized by the serum response factor (SRF), a 67 kDa phosphoprotein which binds as a homodimer to the SRE (Treisman, 1992). The activity of SRF is necessary for the ability of the c-fos SRE to respond to serum stimulation. Experiments have shown that removal of SRF from cell extracts reduces the c-fos transcription to basal levels, while transcription can be restored to maximal levels by addition of exogenous SRF (Norman and Treisman, 1988; Norman et al., 1988; Prywes et al., 1988). Moreover, in vivo depletion of SRF from cell nuclei following antibody microinjection abolishes the response of the SRE to serum stimulation (Gauthier-Rouviere et al., 1991). SRF can interact and cooperate with other protein factors, such as members of ternary complex factor (TCF) family, to optimize the transcriptional induction of c-fos (Price et al., 1996; Hill and Treisman, 1995). We recently reported that adipocyte differentiation of nontransformed murine 3T3T cells represses the expression and nuclear localization of SRF without effecting SP-1 that was used as a control. Repression of SRF nuclear localization thus impaired the availability of SRF to bind to the SRE. We proposed that this mechanism can explain how adipocyte differentiation represses growth factor responsiveness (Ding et al., 1999). On the basis of those findings, this paper evaluates a series of questions. Is the inhibition of SRF nuclear localization during adipocyte differentiation unique or is it a general effect that involves multiple transcription factors? Does loss of proliferative capacity involving other biological processes, such as cellular senescence, also represses the nuclear localization of SRF? Does differentiation of all cell lineages change SRF nuclear localization in a similar manner?
MATERIALS AND METHODS Cell lines, cell cultures and in vivo tissues acquisition Normal human fetal lung fibroblasts (strain WI-38) and murine 3T3T mesenchymal stem cells were used in these experiments. WI-38 fibroblasts were purchased from NIA aging cell culture repository (Coriell Institute) at population doubling levels (PDL) of 16 and 44, respectively (maximum lifespan of 50 population doublings for this culture was reported by the repository). WI-38 cells were cultured in minimal essential medium Eagle (MEM) containing Earle’s salts, supplemented with 2 mM glutamine, 20 mM NaHCO3 and 10% fetal bovine serum at 37°C in 5% CO2/95% air atmosphere. Early passage cells were defined as those that had progressed through 40% of their replicative life span (PDL of 20 to 24), whereas senescent cells were defined as those that had completed >95% of their life span (PDL of 48 to 50). 3T3T cells were routinely cultured at 37°C in 5% CO2/95% air in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum (BCS). Specimens of normal human small bowel and bone marrow were obtained from the UT Surgical Pathology department from waste materials. Induction of adipocyte differentiation To induce predifferentiation growth arrest and subsequent adipocyte differentiation, growing 3T3T cells were dissociated with 0.1% ethylenediamine tetraacetic acid (EDTA) in phosphate-buffered saline (PBS) and plated onto 100-mm ethylene oxide-sterilized bacteriological petri dishes at low density in heparinized DMEM containing 25% (v/v) human plasma as previously described (Hoerl and Scott, 1989). In this medium, most cells become quiescent within 3 to 4 days and subsequently express the nonterminal adipocyte phenotype between days 6-8. For 3T3T cells, the terminal differentiation phenotype is thereafter expressed between days 10 to 15. The extent of differentiation in such cultures was routinely characterized by phase microscopic examination and exceeded 75%. Isolation of nuclear and total cellular proteins Nuclear proteins for western blotting analysis were isolated using the modified method of Dignam (Dignam et al., 1983). After washing cells twice with 4°C PBS (pH 7.4), the cells were harvested in 4°C PBS with a cell scraper. After centrifugation at 500 g for 5 minutes at 4°C, cells were resuspended and incubated for 10 minutes on ice in 4°C hypotonic buffer (1.5 mM MgCl2, 10 mM N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM dithiothreitol (DTT), pH 7.9), the volume of the buffer was proportioned to the number of collected cells. After centrifugation at 25,000 g for 15 minutes at 4°C, pellets were collected. Nuclear proteins were extracted from the pellets. Pellets were washed once with hypotonic buffer and centrifuged at 10,000 g for 15 minutes at 4°C, after that pellets were suspended in ice-cold low-salt buffer (25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT, 20 mM KCl, 20 mM HEPES, pH 7.9). Nuclear protein were released by adding a high-salt buffer (25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT, 1.2 M KCl, 20 mM HEPES, pH 7.9) drop by drop using the half of the volume of low-salt buffer. Samples were incubated on ice for 30 minutes, with frequent smooth mixing. After centrifugation at 25,000 g for 30 minutes at 4°C, the supernatant containing nuclear proteins was collected and stored at −80°C. The total cellular proteins were prepared as previously described (Wang et al., 1996). Briefly, cell monolayers were rinsed twice with 4°C PBS and were then harvested in 4°C PBS. After centrifugation at 500 g for 5 minutes at 4°C, cell were lysed at 4°C for 10 minutes in lysis buffer that contain PBS (pH 7.4), 1% Triton X-100, 0.5% sodium fluoride and 10 mM sodium pyrophosphate. After centrifugation at 30,000 g at 4°C for 10 minutes, total cellular proteins were collected from the supernants and frozen at −80°C. The protein concentrations were measured using the Protein Assay Kit of Bio-Rad and their assay protocol. Indirect immunofluorescence Undifferentiated or differentiated 3T3T cells, or WI-38 fibroblasts (young or senescent) were allowed to attach to slides overnight at 37°C in culture medium. Cells were briefly rinsed with PBS and then fixed for 20 minutes with 4% (w/v) paraformaldehyde in 100 mM sodium phosphate (pH 7.4). Fixed cells were washed in PBS containing 10 mM glycine three times for 5 minutes each, then permeablized for 5 minutes with 1% Nonidet P-40 in PBS plus glycine, and washed as before. Slides were exposed to various dilutions (1:50 to 1:200) of rabbit anti-SRF antibody (Santa Cruz Biotechnology, Inc.) for 60 minutes. Cells were rinsed as before and incubated with fluorescein-conjugated goat anti-rabbit IgG diluted
SRF modulation by senescence and differentiation
1013
1:200 (Santa Cruz Biotechnology, Inc.) for 45 minutes. Slides were washed with three changes of PBS, then coverslips were mounted with mounting medium containing 4′,6-diamidino-2-pheylindole (Vector Laboratories, Inc.). The stained cells were examined and photographed using a confocal laser scanning fluorescence microscope (Zeiss LSM 510). Normal human bone marrow slides were stained following the same procedure, except that slides were further incubated with 100 µg/ml RNAase and 0.05 µg/ml propidium iodide in PBS for 10 minutes before final washes. Western blotting analysis Western immunoblotting procedures were performed as previously described (Wang and Scott, 1994). The primary antibodies used in these experiments include rabbit antibodies against SRF, YY1, C/EBPα, CREB-1, AP-2α, N-Myc and p53, from Santa Cruz Biotechnology, Inc. Protein samples were mixed with sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 minutes, and then separated by electrophoresis in 7.5% to 12% SDS-polyacrylamide gel. After the transfer of proteins onto nitrocellulose membranes, they were incubated for 1 hour at room temperature in the western blocking buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% (v/v) Tween-20, and 1% (w/v) nonfat dry milk. Immunological evaluation was then performed overnight at 4°C in the western blocking buffer containing 1:300 dilution of primary antibodies. The membranes were subsequently washed with western blocking buffer and incubated for 1 hour at room temperature with a peroxidaseconjugated secondary antibody (1:2000 dilution, Sigma). After extensive washing with western blocking buffer, the immunocomplexes on the membrane were visualized using ECL western blotting analysis kit (Amersham Corp.). Immunohistochemistry The formaldehyde-fixed, paraffin-embedded human tissue sections were dewaxed by incubating in xylenes and subsequently rehydrated by processing through a series of washes with decreasing ethanol solution (100%, 95%, 70% and 50%) and then H2O. To quench endogenous peroxidase activity, the sections were incubated in methanol containing 0.3% (v/v) hydrogen peroxide for 30 minutes. Then, the sections were blocked by incubation in PBS containing 3% normal goat serum for 60 minutes at room temperature to prevent nonspecific binding of the antibodies to the tissue. Thereafter, the sections were incubated with primary rabbit anti-SRF antibody (1:100 dilution in PBS containing 1.5% goat serum, Santa Cruz) overnight at 4°C. After this incubation period, the section slides were rinsed with three changes of PBS for 5 minutes each, then exposed to 1:300 dilution of peroxidase-conjugated goat anti-rabbit IgG (Sigma) for 90 minutes at room temperature. After having been rinsed as before, the peroxidase label was developed by exposure to 0.05% (w/v) diaminobenzidine in phosphate buffer containing 0.01% H2O2 for 3 minutes. The slides were washed in PBS, counterstained by hematoxylin (Sigma), and dehydrated by an ethanol/xylene series before coverslips were finally mounted. The specimens were viewed using a standard light microscope (Carl Zeiss).
RESULTS The nuclear localization of SRF is specifically repressed during adipocyte differentiation of 3T3T cells We recently reported that the nuclear localization of SRF is dramatically decreased during the process of adipocyte differentiation of murine 3T3T cells (Ding et al., 1999). This paper expands that analysis to determine whether adipocyte differentiation represses the nuclear localization of multiple
Fig. 1. Western blots comparing the nuclear distribution of seven transcription factors in undifferentiated and differentiated 3T3T cells. Equal amounts of nuclear proteins prepared from growing undifferentiated (RG) and differentiated (NTD) 3T3T cells were subjected to electrophoresis on SDS-polyacrylamide gels and western blotting. Blots were respectively probed with anti-SRF, antiYY1, anti-C/EBPα, anti-CREB-1, anti-AP-2α, anti-N-Myc and antip53, as indicated. Separate assays were performed for each transcription factor and each assay included its own loading control. Heat shock protein 90 (HSP90)was used as the loading control because its expression does not change significantly during adipocyte differentiation.
transcription factors or just SRF. We specifically tested the intracellular localization of six additional transcription factors including YY1, C/EBPα, CREB-1, AP-2α, N-Myc and p53. In undifferentiated proliferating cells, all these factors are predominantly localized in the nucleus. Next, our studies asked if the relative amount of individual transcription factors differs in nuclear extracts of undifferentiated versus differentiated 3T3T cells. Fig. 1 clearly shows that among these transcription factors, SRF is the only factor whose nuclear content is dramatically decreased in differentiated 3T3T adipocytes. In regard to other factors, the amount of YY1, CREB-1, AP-2α, N-Myc and p53 in the nucleus shows no significant difference between differentiated and undifferentiated cells, while that of C/EBPα shows a obvious increase when cells differentiate. This latter finding is consistent with previous reports that the expression of C/EBPα is induced in the late stage of adipocyte differentiation (Cao et al., 1991). Table 1 quantitates and summarizes the data presented in Fig. 1. On the basis of the expression of these factors relative to loading controls in various experiments, the data confirm that in differentiated adipocytes, SRF exhibits a 95% reduction in expression, whereas the other tested factors show significantly less relative reduction in expression in association with differentiation. This suggests that repression of SRF nuclear localization is specific in differentiated adipocytes.
1014
JOURNAL OF CELL SCIENCE 114 (5)
Table 1. Densitometric quantification of the effect of adipocyte differentiation on the nuclear distribution of seven transcription factors Relative optical density Factor
RG
NTD
Ratio NTD/RG
SRF YY1 C/EBPα CREB-1 AP-2α N-Myc p53
6 9 11 7 13 13 7
