HTERT IMMORTALIZES BOVINE LENS EPITHELIAL CELLS AND SUPPRESSES DIFFERENTIATION

THROUGH REGULATION OF THE ERK SIGNALING PATHWAY

Juan Wang1, 4, 5, Hao Feng2, 4, Xiao-Qin Huang2, 4, Hua Xiang1, 6, Ying-Wei Mao1, 7, Jin-Ping Liu3, Qin Yan2, Wen-Bin Liu2, Yan Liu2, Mi Deng2, Lili Gong2,

Shuming Sun2, Chen Luo2, Shao-Jun Liu2, Xuan-Jie Zhang2

Yun Liu2 and David Wan-Cheng Li 1, 2, 3

From the 1Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, NJ 08084, USA; 2College of Life Sciences, Hunan Normal

University, Changsha, Hunan, China 410081; 3Hormel Institute, University of Minnesota, Austin, MN 55912, USA

Running Title: hTERT regulates differentiation via ERK Address correspondence to: David Wan-Cheng Li, The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA. Tel: 507-437-9636; Fax: 507-437-9606; E-mail: dwli1688@hotmail.com. 4 These authors have equally contributed to the present study; 5Current address: Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Genetics and The Penn Diabetes Center, University of Pennsylvania, Philadelphia, PA 19104; 6Current address: State Key Laboratory of Microbial Resources and Center for Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P.R. China; 7Current address: Department of Pharmacology, University of Michigan, Ann Arbor, MI, 48109. Key Words: Telomerase, Lens, Senescence, Differentiation, ERK, MEK, RAF-1, RAS, PKC, PKA, Signal Transduction.

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The functional telomerase complex contains a telomerase reverse transcriptase catalytic subunit (TERT) and a telomerase template RNA (TR). We have previously demonstrated that human telomerase reverse transcriptase catalytic subunit (hTERT) is functionally compatible with a telomerase template RNA from rabbit (rTR). In the present study, we show that hTERT is also functionally compatible with a telomerase template

RNA from bovine (bTR). Introduction of hTERT into bovine lens epithelial cells (BLECs) provides the transfected cells telomerase activity. The expressed hTERT in BLECs supports normal growth of the transfected cells for 108 population doublings (PDs) so far and these cells are still extremely healthy in both morphology and growth. In contrast, the vector-transfected cells display growth crisis after 20 PDs. These cells run into cellular senescence due to shortening of the telomeres and also commit differentiation as indicated by the accumulation of the

JBC Papers in Press. Published on April 22, 2005 as Manuscript M500032200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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differentiation markers, β-crystallin and filensin. hTERT prevents the occurrence of both events. By synthesizing new telomere, hTERT prevents replicative senescence; and through regulation of MEK/ERK, PKC and PKA, and eventual suppression of the MEK/ERK signaling pathway, hTERT inhibits differentiation of BLECs. Our finding that hTERT can suppress RAS/RAF/MEK/ERK signaling pathway to prevent differentiation provides a novel mechanism to explain how hTERT regulates cell differentiation. Telomeres are specialized structures present at the ends of eukaryotic chromosomes, consisting of tandem arrays of highly conserved hexameric TTAGGG repeats in vertebrates (1-2), which are associated with binding proteins such as TRF1 and TRF2 to form a T-loop structure (3). Telomeres have been implicated in stabilizing and protecting linear chromosomes from exonucleolytic degradation, preventing chromosome-to-chromosome fusions and recombination, anchoring chromosomes within the nucleus and assisting the replication of linear DNA (1-2). Telomerase, initially identified in Tetra-hymena (4), is a specialized DNA polymerase that adds telomeric sequences onto chromosome ends, and provides a mechanism to balance the loss of repeats from chromosome ends during cell division (2). Structurally, telomerase is a ribonucleoprotein enzyme complex that contains a catalytic protein subunit, and an essential RNA component (2, 5-8). The catalytic protein subunit contains the telomerase reverse transcriptase activity. In humans, the telomerase reverse transcriptase catalytic subunit (hTERT) is a 127-kD protein (9-12). The encoding

sequence for this catalytic subunit has been cloned in human and also other species (9-14). Apparently, telomerase from these different sources shares both sequence and functional similarity with the reverse transcriptase (15). Recently, the gene encoding the telomerase template RNA has been cloned in 32 different species, and the topological structures of these different telomerase RNAs are well conserved (16). Both in vitro and in vivo reconstitution studies in a number of settings have revealed that hTERT can form a functional complex with human template RNA (hTR) (17-20). Introduction of the human telomerase catalytic subunit (hTERT) into telomerase-negative normal human cell types enabled these cells to obtain elongated telomeres and an extended lifespan (21-27). Whether hTERT is functionally compatible with a template RNA from other vertebrates remains unknown until our recent study in which we have demonstrated that hTERT can form a functional telomerase complex with a rabbit template RNA (28). In the present study, we further demonstrate this functional compatibility between hTERT and a template RNA from bovine. Expression of hTERT in bovine lens epithelial cells (BLEC) results in elongated telomeres and extended lifespan. Besides telomere maintenance, our previous studies have demonstrated that telomerase has other functions beyond telomere synthesis. First, overexpression of hTERT in rabbit lens epithelial cells not only yields telomerase activity but also distinctly suppresses camptothecin-induced apoptosis. The suppression of induced apoptosis by hTERT is associated with its repression of expression of the apoptotic genes including p53 and Bcl-XS, but not linked to telomere synthesis

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(28). Second, when introduced into human lens epithelial cells regardless the presence or absence of the endogenous telomerase activity, hTERT regulates the RB/E2F pathway to accelerate the cell growth rate (29).

In the present study, we report that hTERT displays additional functions beyond telomere synthesis. We demonstrate here that hTERT introduced into BLECs can suppress differentiation. Furthermore, we provide the first evidence that hTERT can regulate the RAS/RAF/MEK/ERK pathway to mediate the suppression of lens epithelial cell differentiation. Thus, our studies provide evidence that hTERT regulates both proliferation and differentiation in eukaryotes.

EXPERIMENTAL PROCEDURES Establishment of Stable Expression Cell Lines—The hTERT expression construct, pCI-hTERT, was kindly provided by Dr. Robert Weinberg (30). The parallel control vector pCI-neo was purchased from Promega. The pCI-neo and pCI-hTERT were introduced into BLECs derived from primary cultures using electroporation with a BTX Electro Cell Manipulator as described before (28-29). Selection of individual stable expression cell clone was conducted in the corresponding medium containing 600 µg/ml G418 (Gibco BRL) for 6 weeks. RNA Preparation and Semiquantitative RT-PCR—Total cellular RNAs were extracted from 100% confluent cultures with Trizol reagent (Life Technologies, Inc.) as previously described (31-32), and 2 µg of total RNAs were used for reverse transcription in a 20 µl reaction with Superscript II (reverse transcriptase, from Life Technologies, Inc.) according to the manufacturer’s protocol. Two µl of the

reverse transcripts were used for PCR. To determine the expression of hTERT, semiquantitative RT-PCR was conducted using β-actin as an internal control. The primers used in this study included: 5’-GTGGGGCGCCCCAGGCACCA-3’ (forward) and 5’-CTCCTTAATGTCAC-GCCGATTTC-3’ (reverse) for the β-actin gene; 5’-GCTGATGAGTGTGTACGTC-G-3’ (forward) and 5’-CTCTTTTCTC-TGCGGAACGT-3’ (reverse) for hTERT. PCR was run 4 cycles with hTERT primers before β-actin primers were added for another 30 cycles. Immunoprecipitation-Linked RT-PCR— The immunoprecipitation-linked RT-PCR was conducted as previously described (28). Briefly, total cellular proteins from both pCI-BLECs and pCI-hTERT-BLECs were extracted using 1 x CHAPS lysis buffer. The anti-hTERT antibody (Alpha Diagnostic) was used to precipitate the hTERT-bTR complex from pCI-hTERT-BLEC stable expression clone. A parallel control precipitation from the pCI-BLEC clone was also conducted. Five hundred µg of cell extracts were mixed with 10 µg anti-hTERT antibody and 100 units of RNasin (Promega). The mixture was incubated at 4°C for 1 hour with gentle shaking, then 50 µl of protein A/G plus agarose (Santa Cruz Biotechnology) was added into the initial reaction mixture and incubated for an additional hour under the same condition. After the incubation, the mixture was centrifuged for 15 minutes at 12,000 rpm at 4°C, and the pellet was washed three times with PBS. Following the final wash, the pellet was processed for RNA isolation using Trizol as described above. Ten µg of E. coli tRNA was added into the sample as carrier before RNA extraction. The isolated RNA was processed for RT-PCR. The primers for bovine telomerase RNA are 5’-

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CTGTGCTTTTGGTTACCGCG-3’ (for-ward) and 5’-TTCCTTTCCCTGCGGTT-ACA-3’ (reverse). Cell Extract Preparation and Telomerase Activity Assay—Cellular extracts from parental BLECs, vector-transfected BLECs (pCI-BLEC) and hTERT-transfected BLECs (pCI-hTERT-BLEC) were prepared in 1 × CHAPS lysis buffer and cleared by centrifugation for 20 min at 4ºC (12,000g). These extracts were assayed for telomerase activity with a PCR-based telomeric repeat amplification protocol (TRAP) (33) using a kit from Intergen. Briefly, telomeric repeats were synthesized onto the oligonucleotide, TS (5’-AATCCGTCGAGCAGAGTT-3’), in an extension reaction containing each cell extract sample and run at 30oC for 30 min. After extension, the products were amplified by PCR with γ-32P-ATP end-labeled TS primer and a downstream TRAP primer mixture (Intergen) for 30 cycles (94oC, 30s, and 59oC, 30s). A 36-base pair internal standard was included for each reaction. At the end of each reaction, 25 µl of the reaction products were analyzed in a 10% polyacrylamide gel. Southern Blot Analysis for Telomere Length—To determine the length of telomeres in both vector- and hTERT-transfected bovine lens epithelial cells, Southern blot analysis was conducted as previously described (34-36). Briefly, genomic DNA samples were extracted from both vector-transfected cells (pCI-BLEC) and hTERT-transfected cells (pCI-hTERT-BLEC). Ten volumes of extraction buffer (10 mM Tris·HCl, pH 8.0; 0.1 M EDTA, pH 8.0; 0.5% SDS; 20 µg/ml pancreatic RNase) were added to the cell pellet which was resuspended by gentle pipetting. The cell suspension was

incubated for 1 hour at 37ºC, then protease K was added to a final concentration of 100 µg/ml. After overnight incubation at 37ºC, phenol extraction was performed for 3 times. The DNA was precipitated, washed, and resuspended in T.E (10 mM Tris·HCl, pH 8.0/1 mM EDTA). The genomic DNA was digested with restriction enzymes Hinf I and Rsa I (Promega) at 37ºC overnight, then precipitated with 2.5 volumes of 100% ethanol in the presence of 20 µg E. coli tRNA as carrier. The DNA fragments were resolved by 0.8% agarose gel electrophoresis at 100V for 4 hours. After depurination by soaking in 0.1 M sodium citrate (pH 3.0) for 30 min and denaturation with 1.5 M NaCl/0.5 M NaOH for 20 min twice, the DNA fragments were neutralized with 1.5M NaCl/0.5M Tris·HCl (pH 7.0), then transferred onto a nitrocellulose membrane in 10x SSC (1.5M NaCl/0.15M sodium citrate). The membrane was pre-hybridized overnight at 40oC in the pre-hybridization buffer and then hybridized with a [γ-32P]-ATP-labeled oligonu-cleotide (TTAGGG)7 probe in the same buffer at 40ºC for 30 h, and washed in moderate stringency condition three times. DNA fragments were visualized by autoradiography. Protein Preparation and Western Blot Analysis—Western blot analysis was conducted as we previously described (37-39). Total protein was extracted from BLECs, pCI-BLECs and pCI-hTERT-BLECs using 300 µl of extraction buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 9.1 mM Na2HPO4, 1.7 mM NaH2PO4, 150 mM NaCl, 100 µg/ml, 30 µl/ml aprotinin with pH adjusted to 7.4). The protein concentrations were determined as described before (40). Fifty or 100 µg of

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total protein from each sample were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Gibco BRL). The protein blots were blocked with 5% milk in TBS (10mM Tris·HCl, pH 8.0, 150mM NaCl) overnight at 4ºC, and then incubated for 1 hour with anti-hTERT, anti-ERK, anti-p-ERK, anti-MEK, anti-phospho-MEK, and anti-β-actin antibodies (Santa Cruz Biotechnology), anti-PKC and anti-PKA antibodies (Pharmingen), anti-β-crystallin antibodies (StressGen) and anti-filensin antibody (Sigma) at a dilution of 1:500 to 2000 (µg/ml) in 5% milk prepared in TBS. The secondary antibody was anti-mouse IgG, anti-rabbit IgG or anti-goat IgG (determined by primary antibodies) at a dilution of 1:1000 to 2000 (Amersham Pharmacia Biotech). Immunoreactivity was detected with an enhanced chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech) according to the company’s instructions.

Overexpression of PKA, PKC and MEK, and Inhibition of PKA, PKC and RAF/MEK/ERK Signaling Pathways with Pharmacological Inhibitor or Dominant Negative Mutants—The expression vectors of pCMV-PKAα and pCMV-MEK were obtained from the Clontech, Palo Alto, California. The PKCα expression vector, pCMV-PKCα and its dominant negative mutant (DNM), pCMV-DNM PKCα were kind gifts of Dr. Zigang Dong (University of Minnesota). The PKA specific inhibitor, H-89 (Ki=48 nM for PKA) was purchased from Calbiochem. Inc. Both vector- and hTERT-transfected BLECs were treated with 250 nM H-89 for 24 hours before the treated cells were harvested for analysis. H-89 does not inhibit other kinases at the concentration used. The RAF and RAS

dominant negative mutants (41-42) were obtained from the Clontech, Palo Alto, California. Both overexpression and DNM constructs were amplified in DH-5α and purified using the Plasmid Maxiprep Kit from Bio-Rad (CAT# 732-6130). Transfection of pCI-BLECs and pCI-hTERT-BLECs was conducted with LipofectamineTM 2000 from the Invitrogen Life Technologies according to the company instruction manual. Expression levels of each construct in the transfected cells were monitored with Western blot analysis. Cell Growth Assay—To determine the cell growth rate, equal number of cells from different cell lines were seeded in 6-well plates at day 0. The medium was replaced every two days, and the cells in each well were collected by trypsinization and counted using hemocytometer in triplicate, in an interval of 24 hours for 5 days.

Senescence Associated-β-Galactosidase Assay—Senescence-associated β-galactosidase (SA−β−Gal) assay was used to determine cellular senescence. Senescent cells express higher level of lysosomal β-Gal activity detectable in the presence of X-Gal at pH 6.0, which forms a local blue precipitate upon cleavage (43). Briefly, 3 x 104 cells of vector-transfected cells (pCI-BLEC) and two clones of hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) at 10 and 20 PDs were plated in slide culture chambers and allowed a total of 48-hr of growth. Then, media were removed and the cells in each chamber were washed with PBS twice, followed by fixation with 3% formaldehyde for 5 min. After fixation, the cells in each chamber were washed with PBS twice and then incubated for 12 hours with fresh SA-β-Gal staining

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solution (1mg/ml X-Gal, 5mM potassium ferricyanide, 5mM potassium ferrocyanide, 2 mM MgCl2 in PBS) (43). For quantitation of senescent cells in each sample, a total of 200 cells were counted in each field and 6 fields in 3 slide chambers were examined. The results were shown in Table 1. Quantitation of the Total Protein And Kinase Activity Levels—For quantitative comparison, some of the x-ray films were analyzed with the automated digitizing system from the Silk Scientific Corporation. Total pixel data were averaged from three experiments after normalization against the background and β-actin.

RESULTS The Establishment of the hTERT-Expression Bovine Lens Epithelial Cell Line—We have previously demonstrated that hTERT is functionally compatible with the telomerase RNA from rabbit (28). To further confirm the functional compatibility between hTERT and other vertebrate TR, we have introduced the hTERT into bovine lens epithelial cells (BLEC) derived from primary cultures. The primary cultures of BLECs were established from intact bovine lens organ as described in the Experimental Procedures. These cells lack detectable telomerase activity as demonstrated by TRAP assay (left lane in Fig. 1C). After introduction of either the vector, pCI-Neo or the hTERT expression construct, pCI-hTERT into BLECs, there cells underwent a 6-week selection in the presence of 600 µg/ml G418. Thereafter, the stable clones either carrying only the vector or expressing hTERT were obtained. Fig. 1A showed the morphology of a 100% confluent culture of one of the hTERT expression line (pCI-hTERT-BLEC-C1).

RT-PCR was used to detect the mRNA expression of hTERT in this line and another line (pCI-hTERT-BLEC-C2). As shown in top panel of Fig. 1B, the 309 bp hTERT DNA band amplified from hTERT mRNA was detected only in the two clones of hTERT-transfected BLECs but not in vector-transfected cells. The expression of hTERT was further confirmed by Western blot analysis as shown in the bottom panel of Fig. 1B. The successful expression of the hTERT in BLECs does not necessarily imply that it is functional since the telomerase requires the correct template RNA for the execution of its function. Therefore, telomerase activity was analyzed for the vector-transfected cells and the two clones of hTERT-transfected cells by TRAP assay. As shown in Fig. 1C, telomerase activity was only detected in the two clones expressing hTERT. Thus, hTERT-expression stable lines (pCI-hTERT-BLEC-C1/-C2) were established.

The Overexpressed hTERT Protein Is Functionally Compatible with the Bovine TR—The presence of telomerase activity in hTERT-transfected cells but not in vector-transfected cells suggests that hTERT is functionally compatible with the telomerase template RNA from bovine. To further confirm the direct interaction between hTERT and bTR, we performed immunoprecipitation-linked RT-PCR. First, we precipitated the cell extracts from both pCI-hTERT-BLEC and pCI-BLEC cells with an anti-hTERT antibody. Then, the precipitated complexes were used for RNA extraction, and the extracted RNA samples were processed for RT-PCR analysis. As shown in Fig. 2B, a predicted DNA band of 296 bp was detected in the RT-PCR reaction with the immunoprecipitated RNA from the pCI-hTERT-BLEC cell line, but not in

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that from the pCI-BLEC cells. Treatment of the precipitated samples with RNase led to degradation of the bTR, and thus, the 296 bp band was not detected (Fig. 2C). Together, these results confirm that hTERT interacts with bTR and this interacting complex is functional. hTERT Supports Normal Growth of pCI-hTERT-BLECs—The parental bovine lens epithelial cells (BLEC), vector-transfected cells (pCI-BLEC) and the two clones of hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) were cultured continuously. After 20 PDs, both the parental and the vector-transfected BLECs displayed extremely slow growth (Fig. 3). In contrast, the two clones of hTERT-transfected BLECs maintain normal growth rates (Fig. 3). So far, the two clones of hTERT-transfected BLECs (pCI-hTERT-BLEC-C1/-C2) have been passaged for 108 and 88 PDs, respectively. They are still in normal growth condition (Fig. 3). hTERT Prevents Cellular Senescence in pCI-hTERT-BLECs—We predicted that the extremely slow growth rate of the pCI-BLEC cells may be due to cellular senescence. To test this possibility, we performed the SA-β-Gal assay, a standard procedure to detect senescent cells. As shown in Fig. 4, the vector-transfected BLEC cells were larger in size, displayed a flattened morphology, and about 60% of these cells were β-Gal positive (Table 1). All of these are typical characteristics of senescent cells. In contrast, the two clones of hTERT-transfected BLEC cells showed no such changes and less than 5% β-Gal positive cells were found (Table 1). These results showed that hTERT could prevent cellular senescence in the hTERT-transfected clones of BLECs.

hTERT Supports Normal Growth of pCI-hTERT-BLEC Cells by Synthesizing Telomeres—To explore the possible mechanism by which hTERT prevents cellular senescence, we examined telomere lengths in vector-transfected Cells (pCI-BLEC) and two clones of hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) at 20 PDs. As shown in Fig. 5, the two clones of hTERT-transfected BLECs displayed a maximal telomere length of 12 kb. In contrast, the vector-transfected BLEC cells had a maximal telomere length of 10 kb, which was 2 kb shorter than that of hTERT-transfected BLEC cells. These results indicate that hTERT introduced into BLECs can synthesize new telomere, which helps to prevent cellular senescence. hTERT Prevents Cell Differentiation in pCI-hTERT-BLEC Cells—The in vivo lens epithelial cells undergo differentiation after limited divisions at the germinal zone (44-47). The differentiated cells express differentiation markers such as β-crystallin and filensin (44-48). Both the parental BLECs and the vector-transfected BLECs after 20 PDs displayed extremely slow growth rate. It is possible that these cells may commit differentiation. To test this possibility, we have examined expression of β-crystallin and filensin at 10 and 20 PDs using Western blot analysis. As shown in Fig. 6A, β-crystallin and filensin were expressed at significant levels at 10 PDs and substantially upregulated at 20 PDs in the parental and the vector-transfected BLEC cells. In contrast, in the two clones of hTERT-transfected BLEC cells, expression of both β-crystallin and filensin were not detected at either 10 or 20 PDs. Inhibition of telomerase activity led to expression of β-

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crystallin and filensin, suggesting the induction of differentiation. These results demonstrated that hTERT expressed in BLECs is able to suppress differentiation. hTERT Represses Activation of ERK 1/2 and MEK1/2—Previous studies (48-58) have shown that MAP kinases especially ERK1/2 kinases are involved in regulation of lens cell differentiation. To examine the possible mechanism by which hTERT prevents lens cell differentiation, we have examined expression and activity of the ERK 1/2 in parental BLECs, vector-transfected cells and the two clones of hTERT-transfected BLECs at 10 and 20 PDs using Western blot analysis. As shown in Fig. 6B, the ERK1 activity was only present in the parental and vector-transfected cells at both 10 and 20 PDs. The ERK2 activity was about 20-fold higher in the parental and the vector-transfected cells than that in the two clones of hTERT-transfected cells. Inhibition of telomerase activity in hTERT-transfected cells resulted in the recovery of the activities of ERK1/2. These results suggest that hTERT mediated suppression of differentiation is associated with its repression of ERK 1/2 activation. Since ERK1/2 is activated by MEK1/2 (59-60), we next examined the expression and activity of MEK1/2 in the parental BLECs, the vector- and the two clones of hTERT-transfected BLECs again at 10 and 20 PDs with Western blot analysis. As shown in Fig. 6C, expression of total MEK1/2 was downregulated in both clones of hTERT-transfected BLECs (pCI-hTERT-BLEC-C1/-C2) by 4- to 8-fold at 10 and 20 PDs compared with that in the parental BLECs and also in the vector-transfected pCI-BLEC cells. The MEK activity was barely detectable in both clones of hTERT-transfected cells.

In contrast, MEK activity was easily detected in both the parental BLECs and the vector-transfected BLECs at either 10 or 20 PDs. Inhibition of telomerase activity led to elevated expression of MEK1/2 and also enhanced activity of these two kinases. Together, these results suggest that hTERT suppresses ERK1/2 through down-regulation of MEK1/2 expression and activity. Suppression of the RAS/RAF/MEK/ERK Signaling Pathway Is Necessary for hTERT to Prevent Differentiation of BLECs—To demonstrate that hTERT-mediated suppression of the MEK/ERK signaling pathway is necessary for hTERT to prevent differentiation of BLECs, we have introduced the dominant negative mutant RAF and RAS, which are known to interfere with activation of the MEK/ERK kinases (41-42), into both vector-transfected cells (pCI-BLEC) and hTERT-transfected cells (pCI-hTERT-BLEC-C1). As shown in Fig. 7, these mutants not only displayed interference with activation of MEK/ERK kinases but also greatly attenuated expression of β-crystallin and filensin in both vector- and hTERT-transfected BLECs. To further confirm that hTERT suppresses differentiation through MEK/ERK pathway, we overexpressed MEK in both vector and hTERT-transfected cells. Previous studies have shown that overexpression of MEK activates ERK1/2 (61-63). Overexpression of MEK1 in both vector- and hTERT-transfected cells not only activates ERK1/2 and also promotes differentiation as reflected by the expression of β-crystallin and filensin (Fig. 8). However, overexpression of MEK1 did not affect the senescence cell levels of the vector- and hTERT-transfected cells (Table 1). Thus, overexpression of MEK only overrides the

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hTERT suppression of differentiation. Together, our results showed that hTERT prevents differentiation of BLECs through suppression of the RAS/RAF/MEK/ERK signaling pathway. hTERT Modulates Expression of PKAα and PKCα which Enhances Suppression of MAP Kinases and Differentiation—Since protein kinase A (PKA) and protein kinase C (PKC) are involved in regulation of MAPK (63-64), we next examined whether hTERT also regulates these kinases. Western blot analysis revealed that hTERT modulated their expression. As shown in Fig. 9, expression of the catalytic subunit of PKA (PKAα) was upregulated about 2-fold in the hTERT-transfected cells than that in vector-transfected cells. In contrast, the expression of the catalytic subunit of PKC (PKCα) was downregulated about 4-fold in hTERT-transfected cells than that in vector-transfected cells. Inhibition of the telomerase activity in hTERT-expression cells largely reversed the expression patterns of these two kinases to that found in the vector-transfected cells. These results suggest that hTERT may modulate additional upstream kinases to enhance the suppression of the RAS/RAF/MEK/ERK signaling pathway. To confirm that PKA could negatively regulate ERK1/2 and differentiation in BLECs, we overexpressed PKAα or inhibited the activity of this kinase in both vector- and hTERT-transfected BLECs. As shown in Fig. 10, overexpression of PKAα substantially attenuated ERK1/2 activation and largely inhibited expression of the two differentiation markers, β-crystallin and filensin in vector-transfected cells. In hTERT-transfected cells, because PKAα was expressed in high level and hTERT suppressed both ERK1/2 activation and differentiation, the

effect of overexpressing PKA was masked. On the other hand, inhibition of PKA activity by H-89, a specific PKA inhibitor lead to activation of ERK1/2 and expression of the two differentiation markers in the hTERT-transfected cells. These results show that PKA can negatively modulate MAP kinases and differentiation in BLECs and hTERT modulates PKA to enhance suppression of ERK1/2 and differentiation. To demonstrate that PKC can positively modulate MAP kinases and promotes differentiation, we also overexpressed PKCα or blocked its activity by dominant negative mutant (DNM). As shown in Fig. 11, overexpression PKCα in hTERT-transfected BLECs induced activation of ERK1/2 and expression of β-crystallin and filensin. In contrast, inhibition of PKC activity by DNM PKCα suppressed both ERK1/2 activation and expression of the differentiation markers in the vector-transfected cells. Such results indicate that hTERT can negatively modulate PKC to enhance the suppression of the ERK signaling pathway and differentiation in BLECs.

DISCUSSION

In the present study, we have demon-strated the followings. 1) hTERT is functionally compatible with a telomerase template RNA from bovine; 2) hTERT when expressed in bovine lens epithelial cells elongates telomeres of the transfected cells to extend proliferative lifespan and to prevent replicative senescence; 3) hTERT prevents differentiation of the transfected cells; 4) hTERT also suppresses the RAS/RAF/MEK/ERK pathway, and suppression of this pathway is necessary for hTERT to prevent differentiation of BLECs; and 5) hTERT modulates expression of PKA and PKC to enhance

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the suppression of the RAS/RAF/MEK/ ERK pathway and thus the inhibition of differentiation (Fig. 12). hTERT can Function with the Bovine Template RNA—The minimal function core of telomerase contains a reverse transcriptase catalytic subunit (TERT) and a template RNA (TR) (2). Previous studies have shown that human telomerase activity can be reconstituted from these two components either in vitro or in vivo. The first system was reconstituted with partially purified, micrococcal nuclease-treated 293 cell extracts and recombinantly synthesized human template RNA (17). Later, reconstitution was conducted with in vitro transcribed and translated hTERT and either co-synthesized hTR or isolated hTR in the presence of rabbit reticulocyte lysate (18-19). More recently, reconstitution was accomplished with a GST-hTERT fusion protein and hTR co-expressed in yeast (20). In all of these cases, hTERT uses hTR as a functional template. The hTERT has also been expressed in a variety of human cell lines and the expressed hTERT is functional (21-27). This is not surprising since in these cases the hTERT again uses the endogenous human template RNA. Whether the hTERT can use a template RNA from other species had not previously been demonstrated until our recent studies in which we showed that hTERT is functionally compatible with a rabbit template RNA (28). In the present study, hTERT was introduced into bovine lens epithelial cells, which lacks detectable telomerase activity. TRAP assay showed that the exogenous hTERT is functionally compatible with the endogenous bovine TR. Immunoprecipitation-linked RT-PCR confirmed that the expressed hTERT indeed forms a complex with bTR. From

these results and our previous studies, we conclude that hTERT can use non-human TR to form a functional enzyme. Such compatibility largely depends on the conservation of the template RNA (65). In this regard, it is interesting to mention that replacement of the endogenous yeast TR with human TR does not impair yeast growth (66). Whether hTERT can replace endogenous yeast TERT remains to be elucidated.

hTERT Prevents Cellular Senescence through Maintenance of Telomere Length—Cellular or replicative senescence is a process of terminal growth cessation and morphological change displayed by normal cells after they have undergone a finite number of population doublings in vitro (67). Cells undergoing replicative senescence remain adherent to the growth surface and metabolically active for an extended period of time following cessation of proliferation (68). The proliferative potential of a given cell population in culture is directly correlated with the number of prior cell doublings in vivo (67). Two theories have been proposed regarding the senescence basis: the error/genetic damage and the programmed control (70-71). In the programmed control theory, it is considered that a genetic program becomes activated or manifested at the end of the proliferative lifespan of a normal cell, causing the characteristic morphological changes and growth arrest (70-71). Telomere length and telomerase activity can be part of the genetic program. In the absence of telomere elongation, telomeres shorten with each cell division (72). In each round of chromosome replication, telomeres typically lose ~150 base pairs of nucleotide sequence at the 5' end of a DNA molecule. Thus, an increasing cell division number is usually accompanied

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by declining telomere length. Such DNA replication-dependent loss of telomere length seems to be a mitotic clock for counting the number of cell divisions and signaling cellular senescence (73). In our present study, we have shown that the telomere length becomes shortened during division of bovine lens epithelial cells. If the decreased telomere length is not compensated with new telomere synthesis as found in the case of vector-transfected BLECs, the cells then run into senescence (Fig. 4). Telomerase activity is present in germ cells and in most tumor cells but absent in most somatic cells (33). Like most somatic cells, the lens epithelial cells isolated from adult bovine lenses, lack detectable telomerase activity (Fig. 1C). These cells, when cultured under in vitro conditions, can only be passaged for a limited number of generations (approximately 20 PDs). Introduction of the hTERT into these lens epithelial cells confers the transfected cells with telomerase activity (Fig. 1C). Moreover, the two clones of hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) have been passaged for more than 108 and 88 PDs, respectively, and they are still growing normally (Fig. 3). However, the vector-transfected cells display extremely slow growth rate after 20 PDs (Fig. 3). SA-β-Gal analysis of both the vector- and the hTERT-transfected BLEC cells after 20 PDs revealed that approximately 60% of the vector-transfected BLECs have undergone cellular senescence (Fig. 4 and Table 1). Thus, hTERT prevents cellular senescence of the BLEC cells with telomerase activity. The presence of telomerase activity allows hTERT expression cells to carry out new synthesis of telomeres as reflected by the fact that the telomeres in hTERT-transfected cells is about 2 kb longer than that in the

vector-transfected cells. Our results are consistent with numerous earlier studies where the hTERT has been overexpressed in a variety of human cell lines (21-27). In all cases, the hTERT-transfected cells have extended telomere length and lifespan to variable degrees. hTERT Prevents Cell Differentiation through Regulation of the RAF/MEK/ERK Pathway—It is now becoming more clear that telomerase, besides synthesis of telomere, also bears other functions (74). One of such functions is its involvement in regulation of differentiation. Earlier studies have shown that telomerase activity decreases as cells differentiate during brain development (75-76), suggesting a possible role for this enzyme in the differentiation process. Treatment of cells with telomerase antisense RNA (77) can induce differentiation, indicating that telomerase activity may execute control over the proliferation/ differentiation decision. Similar observations have been reported in PC 12 cells (77-78). In the present study, we demonstrate that both parental and vector-transfected BLECs after 10 PDs commit differentiation as reflected by exclusive expression of two differentiation markers, β-crystallin and filensin. When most of these cells become senescent, the differentiation is further enhanced as reflected by the elevated expression of the differentiation markers (Fig. 6). In contrast, in hTERT-expression cells, expression of β-crystallin and filensin was undetectable, suggesting that hTERT suppresses differentiation of the transfected cells. To further confirm the anti-differentiation function of hTERT, telomerase activity in the pCI-hTERT-BLECs was inhibited and expression of the differentiation markers, β-crystallin and filensin was examined. When

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telomerase activity is inhibited, the hTERT-expression cells are forced to enter differentiation status as reflected by expression of β-crystallin and filensin. Thus, hTERT can suppress differentiation of lens epithelial cells. What is the possible mechanism by which hTERT prevents differentiation of BLECs ? Recent studies (48-58) have revealed that the MAP kinases, especially ERK1/2, mediate lens differentiation by different factors. For example, FGF-induced cell proliferation and differentiation require participation of ERK MAP-kinases. ERK activity is found to be necessary and sufficient to induce cell proliferation and differentiation in rat lens epithelia transplants (48). In transgenic mice overexpressing a truncted FGF receptor R3, down-regulation of ERK1/2 phosphorylation (activity) is associated with delayed lens fiber cell differentiation (51). Based on these observations, we hypothesize that hTERT may prevent differentiation of BLECs by regulating MAP kinase pathway. Indeed, when the activity and expression of ERK/MEK kinases in both vector- and hTERT-transfected cells were analyzed with Western blot, it was found that activation of ERK 1 is completely suppressed and ERK2 activity is substantially down-regulated in hTERT-expression cells compared with those in vector-transfected cells. Comparative analysis of the upstream activation kinases, MEK1/2, reveals that in hTERT expression cells, both expression and activity of MEK1/2 are significantly downregulated in hTERT-expression cells than that in the vector-transfected cells or in the parental BLECs undergoing similar PDs (both 10 and 20 PDs). Thus, the suppression of BLEC differentiation by hTERT is associated with the down-regulation of

MEK/ERK expression and activation. To determine that hTERT-mediated repression of MEK/ERK pathway leads to suppression of differentiation of BLECs, dominant negative mutant RAF-1 (blocking activation of MEK1/2, 42) and dominant negative RAS (interfering with activation of RAF-1, 41) were introduced into both vector- and hTERT-transfected cells. These mutants greatly attenuate the activation of MEK1/2 and ERK1/2, and substantially suppress expression of β-crystallin and filensin in vector-transfected cells (Fig. 7). On the other hand, overexpression of MEK in the hTERT-expression cells induces both activation of ERK1/2 and also differentiation (Fig. 8). However, overexpression of MEK does not seem to interfere with the senescence process of the vector- and hTERT-transfected cells (Table 1). Clearly, the enforced activation of ERK1/2 overrides the suppression of differentiation by hTERT. Thus, our results demonstrate that hTERT prevents differentiation of BLECs through suppression of the RAS/RAF/MEK/ERK signaling pathway.

How could the RAS/RAF/MEK/ERK signaling pathway is activated in vector-transfected BLECs but not in hTERT-transfected BLECs ? It has been shown that telomere shortening as found in vector-transfected BLECs could trigger a response, which bears the hallmark of DNA damage response (79-80). In either case, the signal is transmitted through the ATM and ATR kinases, which activate the tumor suppressor, p53 (79-84). Activation of p53 or constitutive expression of p53 has been shown to activate the RAS/RAF/MEK/ERK signaling pathway through activators upstream RAS (85). A recent study suggests that in response to DNA damage, activated p53 induces expression of

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growth factors such as heparin-binding epidermal growth factor-like growth factor (HB-EGF) which then activates the RAS/RAF/MEK/ERK signaling pathway (64). In hTERT-transfected BLECs, hTERT prevents telomere shortening (Fig. 5), and thus can suppress p53-dependent activation of the RAS/RAF/MEK/ERK signaling pathway. Furthermore, hTERT may also suppress the RAS/RAF/MEK/ERK signaling pathway through modulation of PKA and PKC as discussed below. In the present study, we have shown that hTERT also exerts positive control on PKAα expression (Fig. 9). The hTERT-mediated positive regulation of PKAα can enhance the suppression of ERK signaling pathway and prevention of differentiation as demonstrated from two experiments: overexpression of PKAα and inhibition of PKAα activity in vector- and hTERT-transfected BLECs. Overexpression of PKAα substantially inhibits activation of ERK1/2 and largely prevents expression of the two differentiation markers, β-crystallin and filensin in vector-transfected BLECs (Fig. 10). In contrast, inhibition of PKAα activity in hTERT-transfected BLECs induces substantial activation of ERK1/2 and promotes clear expression of β-crystallin and filensin (Fig. 10). Our observations that PKA negatively modulates MAP kinases in BLECs are consistent with a previous study where PKA has been shown to negatively regulate the RAS/RAF/MEK/ ERK pathway through Raf-1 (61). Similarly, hTERT-mediated negative regulation on PKCα expression leads to further suppression of ERK signaling pathway and prevention of differentiation. This is also derived from the results of another two experiments: PKCα overexpression and inhibition in vector- and hTERT-transfected BLECs

(Figs. 9 and 11). Our results that overexpression of PKCα promotes activation of ERK1/2 while inhibition of PKC activity by DNM PKC suppresses the ERK1/2 activation are consistent with the earlier studies in which PKC was found to positively modulate the ERK signaling pathway at multiple sites including RAS (87-88), RAF-1 (89) and other downstream kinases (90-92). Overexpression of PKCα promotes differentiation not only in BLECs as shown in the present study but also in other lens epithelial cells where PKC is found to promote lens cell differentiation through modulation of multiple targets such as intermediate filament, gap-junction and intrinsic membrane proteins (58, 93-96). Taken together, hTERT suppresses the RAS/RAF/MEK/ERK signaling pathway by repressing DNA damage response, and also by differentially modulating the upstream kinases, PKA and PKC. How could suppression of ERK kinases lead to prevention of differentiation ? It is well known that the ERK kinases can phosphorylate multiple transcription factors (59-60, 92). Phosphorylation of these transcription factors has profound effects on their functions. For example, MafA is involved in regulation of lens differentiation (97-100). Phosphorylation of MafA at Ser-14 and Ser-65 by ERK2 kinase enhances its transcriptional activity and therefore, promotes lens specific differentiation (49). In summary, suppression of ERK activa-tion by hTERT results in changed transcriptional activities of multiple transcription factors. As a result, hTERT can suppress expression of differentiation related genes such as the ones encoding β-crystallin and filensin, and therefore, execute the prevention of differentiation status (Fig. 12).

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ACKNOWLEDGEMENTS

We thank Dr. Robert Weinberg for the hTERT expression vector, pCI-hTERT. Dr. Zigang Dong for the PKCα expression vector and the DNM PKC construct. This study is partially supported by the NIH/NEI grant, the Hormel Foundation, the Lotus Scholar Program Funds from the Ministry of Hunan Province Government and also Hunan Normal University.

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FOOTNOTE

The abbreviations used are: Bovine lens epithelial cells, BLEC; Phosphate-buffered saline, PBS; protein kinase A, PKA; the catalytic subunit of PKA, PKAα; protein kinase C, PKC; the catalytic subunit of PKC, PKCα; Ras-associated factor-1, RAF-1; Senescence-associated-β-galactosidase assay, SA-β-Gal assay; SDS-polyacrylamide gel electrophoresis, SDS-PAGE; telomeric repeat amplification protocol, TRAP; telomerase reverse transcriptase, TERT; human telomerase reverse transcriptase, hTERT; telomerase RNA, TR; bovine telomerase RNA, bTR; rabbit telomerase RNA, rTR; Extracellular receptor-activated protein kinases 1 and 2, ERK1/2; Mitogen-activated protein kinases, MAPK; MAPK kinases 1/2, MAPKK 1/2 or MEK 1/2.

FIGURE LEGENDS Fig. 1. Establishment of the stable hTERT-expression BLEC line. The eyeballs were removed from 2-year-old bovines within 2-hours of sacrifice and the lenses were carefully dissected. The anterior lens capsule with adherent lens epithelium was removed and cultured in 60 mm Petri dishes in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) with 10% fetal bovine serum (FBS) and 50 units/ml penicillin and streptomycin. After confluence, the primary cultures were passaged twice before used for transfection. Transfections were conducted using electroporation (28-29). Thirty-six hours after transfection, G418 (600 µg/ml) was added into the culture medium for selection. After a six-week selection, the individual stable clones were obtained. A. The 100% confluent culture from one of the stable lines of hTERT-transfected BLEC cells (pCI-hTERT-BLEC-C1). Bar: 5 micrometer. B. Top panel: RT-PCR to detect the mRNA expression from the exogenous hTERT. Total RNA was extracted with Trizol Reagent (Life Technologies, Inc) from the vector-transfected BLECs (pCI-BLEC) and tow clones of hTERT-transfected BLECs (pCI-hTERT-BLEC-C1/-C2), and RT-PCR was conducted as described in the Experimental Procedures. Note that only the hTERT-transfected BLECs (pCI-hTERT-BLEC-C1/-C2) contain the hTERT mRNA as reflected by an amplified DNA band of 309 bp. The top is the internal control band of 540-bp amplified from the β-actin mRNA. Middle and bottom panels: Western blot analysis of hTERT protein (middle panel) and β-actin (bottom panel) in vector-transfected cells (pCI-BLEC) and hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2). One hundred micrograms of total proteins extracted from vector-transfected cells (pCI-BLEC) and hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) were resolved in 6% SDS-PAGE and analyzed with antibodies against human TERT and β-actin as described in the Experimental Procedures. Note that only the hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) contain the hTERT protein (middle pane). C. Demonstration that the hTERT-transfected cells display telomerase activity. Telomerase activity in parental BLEC (lane 1 from the left), pCI-BLEC (lane 2 from the left) and two clones of pCI-hTERT-BLEC cells (lanes 3 and 4 from the left) was determined with the TRAP assay as described in the Experimental Procedures.

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Fig. 2. Immunoprecipitation-linked RT-PCR to demonstrate that hTERT can form a functional complex with bovine telomerase template RNA (bTR). Immunoprecipitation-linked RT-PCR was conducted as described in the Experimental Procedures. A. RT-PCR with total RNA directly isolated from both pCI-BLEC and pCI-hTERT-BLEC-C1. B. RT-PCR with RNAs isolated from immunoprecipitated samples with the anti-hTERT antibody. C. RT-PCR with RNAs isolated from immunoprecipitated samples with the anti-hTERT antibody, the precipitated samples were treated with RNase (10 µg/reaction) for one hour before extraction of the template RNA. Fig. 3. Expression of hTERT in BLECs supports growth of the transfected cells. The parental BLEC at 20 PDs, the vector-transfected BLEC (pCI-BLEC) at 20 PDs after G418 selection, the established clone 1 of hTERT-transfected BLECs (pCI-hTERT-BLEC-C1) at 20 and 108 PDs after G418 selection, and the established clone 2 of hTERT-transfected BLECs (pCI-hTERT-BLEC-C2) at 20 and 88 PDs after G418 selection were plated in 6-well plates at 2.5 x 104 cells per well. The medium was replaced every two days, and the cells in each plate were counted in triplicate every 24 hours for a continuous 5 days. Note that while both clones of hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) keep normal growth for 108 and 88 population doublings, respectively so far, the parental and vector-transfected BLECs run into growth crisis. Fig. 4. Analysis of cellular senescence in vector-transfected cells (pCI-BLEC, A) and hTERT-transfected cell (pCI-hTERT-BLEC-C1, B). Both vector-transfected cells (pCI-BLEC) and both clones of hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) at 20 PDs after establishment were plated in slide culture chamber at 2 x 104 cells per chamber and allowed a total of 48-h growth. Then, media were removed and the cells in each chamber were washed with PBS twice, followed by fixation with 3% formaldehyde for 5 min. After fixation, the cells in each chamber were washed with PBS twice and then incubated with fresh senescence-associated β-Gal staining solution for 12 hours. The results were either recorded with phase contrast micrograph (shown here) or quantitated (shown in Table 1). The senescent cells were stained blue. Bar: 10 micrometer.

Fig. 5. hTERT prevents telomere shortening in pCI-hTERT-BLECs. The genomic DNA samples isolated from both vector-transfected cells (pCI-BLEC) and the two clones of hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) were digested with HinfI and RsaI, resolved in 0.8% agarose gel and then processed as described in the Experimental Procedures. Note that the vector-transfected BLECs have a maximal telomere length of 10 Kb. In contrast, the two clones of hTERT-transfected BLECs (pCI-hTERT-BLEC-C1/-C2) have a maximal telomere length of 12 kb. Fig. 6. hTERT prevents differentiation of BLECs and suppresses activation of the MEK/ERK signaling pathway. A. hTERT suppresses expression of two differentiation markers, β-crystallin and filensin. The parental bovine lens epithelial cells (BLEC), the vector-transfected cells (pCI-BLEC), and the two clones of hTERT-transfected cells (pCI-hTERT-BLEC-C1/-C2) were grown in DMEM at 10 and 20 PDs to 100% confluence. Inhibition of hTERT activity was conducted as previously described (29).

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Different cell samples were harvested for extraction of total proteins. One hundred micrograms of total proteins from each sample were resolved in 10% SDS-PAGE and analyzed with antibodies against β-crystallin, filensin and β-actin as described in the Experimental Procedures. Note that in the parental (BLEC) and vector-transfected cells (pCI-BLEC), expression of the two differentiation markers, β-crystallin and filensin, was clearly detected at 10 PDs and substantially enhanced at 20 PDs. In contrast, in the two clones of hTERT-expression cells at either 10 or 20 PDs, expression of β-crystallin and filensin was completely suppressed. Inhibition of telomerase activity forces entrance of the hTERT expression cells into differentiation status as reflected by the expression of the two differentiation markers, β-crystallin and filensin. B. Suppression of activation of ERK1/2 by hTERT. One hundred micrograms of total proteins from each sample as described above in Fig. 6A were resolved in 10% SDS-PAGE and analyzed with antibodies against total ERK1/2 (T-ERK1/2), or phospho-ERK1/2 (p-ERK1/2, activated form), or β-actin as described in the Experimental Procedures. Note that in the two clones of hTERT-expression cells at either 10 or 20 PDs, activation of ERK1 was completely suppressed and the activity of ERK 2 was distinctly down-regulated although the total proteins for both kinases are hardly changed in different types of cells. Inhibition of hTERT activity abolishes the suppression of ERK1/2 activation. C. Suppression of expression and activation of MEK1/2 by hTERT. One hundred micrograms of total proteins from each sample as described above in Fig. 6A were resolved in 10% SDS-PAGE and analyzed with antibodies against total MEK1/2 (T-MEK1/2), or phospho-MEK1/2 (p-MEK1/2, activated form), or β-actin as described in the Experimental Procedures. Note that in both clones of hTERT-expression cells, expression of MEK1/2 was substantially down-regulated and the activity of MEK1/2 was barely detectable compared with those in parental BLECs and the vector-transfected cells at both 10 and 20 PDs. Inhibition of hTERT activity abolishes the suppression of MEK1/2 activation. Fig. 7. hTERT-mediated suppression of the MEK/ERK pathway leads to prevention of differentiation by hTERT. Both vector-transfected cells (pCI-BLEC) and hTERT-transfected cells (pCI-hTERT-BLEC-C1) at 18 PDs were further transfected with pCMV vector (lanes 1 and 2 from the left) or with the dominant negative mutant RAS (lanes 3 and 4 from the left, 41) or with the dominant negative mutant RAF-1 (lanes 5 and 6 from the left, 42) and cultured in DMEM in the presence of 600 µg/ml G418 for 48 hours before they were harvested for extraction of total proteins. One hundred micrograms of total proteins from each sample were resolved in 10% SDS-PAGE and analyzed with antibodies against total ERK1/2 (T-ERK1/2), or phospho-ERK1/2 (p-ERK1/2, activated form); total MEK1/2 (T-MEK1/2), or phospho-MEK1/2 (p-MEK1/2, activated form); β-crystallin, filensin or β-actin as described in the Experimental Procedures. Note that in vector-transfected cells, expression of the dominant negative mutant RAS or RAF-1 substantially suppresses activation of MEK1/2 and ERK1/2 and expression of β-crystallin and filensin. Fig. 8. Overexpression of MEK overrides hTERT suppression of activation of the MEK/ERK signaling pathway and differentiation of BLECs. Both vector-transfected cells (pCI-BLEC) and hTERT-transfected cells (pCI-hTERT-BLEC-C1) at 18 PDs were

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further transfected with pCMV vector (lanes 1 and 2 from the left) or with the MEK1 expression vector, pCMV-MEK (lanes 3 and 4 from the left) and cultured in DMEM in the presence of 600 µg/ml G418 for 48 hours before they were harvested for extraction of total proteins. One hundred micrograms of total proteins from each sample were resolved in 10% SDS-PAGE and analyzed with antibodies against total ERK1/2 (T-ERK1/2), or phospho-ERK1/2 (p-ERK1/2, activated form); total MEK1/2 (T-MEK1/2), or phospho-MEK1/2 (p-MEK1/2, activated form); β-crystallin, filensin or β-actin as described in the Experimental Procedures. Note that in hTERT-transfected cells, expression of the MEK1 induces substantial activation of MEK1/2 and ERK1/2, and expression of β-crystallin and filensin, supporting that hTERT suppresses differentiation of BLECs through repression of the MEK/ERK signaling pathway. Fig. 9. hTERT regulates protein kinase A (PKA) and protein kinase C (PKC). The parental bovine lens epithelial cells (BLEC), the vector-transfected cells (pCI-BLEC), and the hTERT-transfected cells (pCI-hTERT-BLEC-C1) were grown in DMEM at 18 PDs to 100% confluence. Inhibition of hTERT activity was conducted as previously described (29). Different cell samples were harvested for extraction of total proteins. One hundred micrograms of total proteins from each sample were resolved in 10% SDS-PAGE and analyzed with antibodies against PKAα, PKCα and β-actin as described in the Experimental Procedures. Note that in hTERT-expression cells, expression of PKAα was up-regulated 2-fold and PKCα down-regulated 4-fold in comparison with expression of the same kinases in parental BLECs and vector-transfected BLECs. Inhibition of telomerase activity leads to significant recovery of the expression patterns of the two kinases as those found in the parental and the vector-transfected cells. Fig. 10. Demonstration that PKA negatively regulate MAP kinases activity and Differentiation in BLECs. Both vector-transfected cells (pCI-BLEC) and hTERT-transfected cells (pCI-hTERT-BLEC-C1) at 18 PDs were further transfected with pCMV vector (lanes 1 and 2 from the left) or with the PKAα expression vector, pCMV-PKAα (lanes 3 and 4 from the left) or treated with 250 nM H-89 for 48 hours ((lanes 5 and 6 from the left). The double transfected cells were cultured in DMEM in the presence of 600 µg/ml G418 for 48 hours before they were harvested for extraction of total proteins. One hundred micrograms of total proteins from each sample were resolved in 10% SDS-PAGE and analyzed with antibodies against total ERK1/2 (T-ERK1/2), or phospho-ERK1/2 (p-ERK1/2, activated form); PKAα, β-crystallin, filensin or β-actin as described in the Experimental Procedures. Note that in vector-transfected cells, overexpression of PKAα substantially suppresses activation of ERK1/2, and expression of β-crystallin and filensin. In contrast, inhibition of PKA activity in hTERT-transfected cells induces activation of ERK1/2 and differentiation of BLECs as reflected by expression of β-crystallin and filensin. These results support that hTERT-mediated positive regulation of PKAα enhances its suppression of the MEK/ERK signaling pathway and BLEC differentiation. Fig. 11. Demonstration that PKC positively regulate MAP kinases activity and Differentiation in BLECs. Both vector-transfected cells (pCI-BLEC) and hTERT-transfected cells (pCI-hTERT-BLEC-C1) at 18 PDs were further transfected with pCMV

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vector (lanes 1 and 2 from the left) or with the PKCα expression vector, pCMV-PKCα (lanes 3 and 4 from the left) or with the dominant negative mutant PKCα construct, pCMV-DNM PKCα (lanes 5 and 6 from the left) and cultured in DMEM in the presence of 600 µg/ml G418 for 48 hours before they were harvested for extraction of total proteins. One hundred micrograms of total proteins from each sample were resolved in 10% SDS-PAGE and analyzed with antibodies against total ERK1/2 (T-ERK1/2), or phospho-ERK1/2 (p-ERK1/2, activated form); PKCα, β-crystallin, filensin or β-actin as described in the Experimental Procedures. Note that in hTERT-transfected cells, overexpression of PKCα causes substantial activation of ERK1/2, and also induces expression of β-crystallin and filensin. In contrast, inhibition of PKC activity in vector-transfected cells substantially suppresses activation of ERK1/2 and expression of β-crystallin and filensin. These results support that hTERT-mediated negative regulation of PKCα also enhances its suppression of the MEK/ERK signaling pathway and BLEC differentiation. Fig. 12. Diagram to show a novel mechanism for hTERT to regulate BLEC differentiation. hTERT, likely by repressing the DNA-damage response, suppresses the upstream signaling components, RAS and RAF-1, leading to down-regulation of expression and activity of the downstream kinases, MEK1/2, which then suppresses ERK1/2. Suppression of ERK1/2 results in the changes in the transactivities of multiple transcription factors such as Maf A. Such changes lead to suppression of expression of the differentiation related genes and prevention of differentiation by hTERT. Besides, hTERT can also positively regulates expression of PKA and negatively control expression of PKC to further enhance the suppression of the RAS/RAF/MEK/ERK signaling pathway and to prevent differentiation. It may be possible that hTERT-mediated regulation of PKA and PKC could directly contribute to prevention of differentiation through other targets. Up-arrow stands for up-regulation; down-arrow on the side of the signaling components represents down-regulation.

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Table 1. Comparison of the Senescent Cell Levels inVector- and hTERT-Transfected Bovine Lens Epithelial Cells*

Cell Types Senescent (Blue) Cells (%)

apCI-BLEC-10 0

apCI-hTERT-BLEC-C1-10 0

aPCI-hTERT-BLEC-C2-10 0

bpCI-BLEC-20 60 + 4.8

bpCI-hTERT-BLEC-C1-20 3 + 1.2

bPCI-hTERT-BLEC-C2-20 2.5 + 1.5

bpCMV-MEK1/pCI-BLEC-20 58 + 6.6

bpCMV-MEK1/pCI-hTERT-BLEC-C1-20

2.5 + 1.6

bpCMV-MEK1/pCI-hTERT-BLEC-C2-20

2.5 + 1.0

*Vector-transfected cells, pCI-BLEC and two clones of hTERT-transfected cells, pCI-hTERT-BLEC-C1 and pCI-hTERT-BLEC-C2, at 10 PDs (a) or 20 PDs (b) after establishment were plated inslide culture chamber at 2 x 104 cells per chamber and allowed atotal of 48-h growth. Then, media were removed and the cells ineach chamber were washed with PBS twice, followed by fixationwith 3% formaldehyde for 5 min. After fixation, the cells in eachchamber were washed with PBS twice and then incubated withfresh senescence-associated b-Gal staining solution for 12 hours.The results were recorded with phase contrast micrograph. Thesenescent cells were stained blue. A total of 200 cells were countedin each field and 6 fields in 3 slide chambers were examined.

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Xuan-Jie Zhang, Yun Liu and David Wan-Cheng LiYan, Wen-Bin Liu, Yan Liu, Mi Deng, Lili Gong, Shuming Sun, Chen Luo, Shao-Jun Liu,

Juan Wang, Hao Feng, Xiao-Qin Huang, Hua Xiang, Ying-Wei Mao, Jin-Ping Liu, Qinthrough regulation of the ERK signaling pathway

hTERT immortalizes bovine lens epithelial cells and suppresses differentiation

published online April 22, 2005J. Biol. Chem. 

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