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作者：Alison M.Devlin, NicolasSolban, SandraTremblay, JolantaGutkowska, WalterSchürch, Sergei N.Orlov, RichardLewanczuk, PavelHamet, JohanneTremblay作者单位：1 Laboratory of Cellular Biology of Hypertension,Centre de Recherche, Centre Hospitalier de l‘Université deMontréal, University of Montreal, Montreal, Quebec H2W 1T and Department of Endocrinology, University ofAlberta, Edmonton, Alberta, Canada T6G 2S2
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; Y) ?0 M/ k; V: T: t# N 【摘要】; X/ _+ W! h, m: a' E* | A/ d3 b
Werecently identified a novel calcium-regulated gene, HCaRG,that is highly expressed in the kidney and maps to a chromosomal locusdetermining kidney weight in rats. The mRNA levels of HCaRG negatively correlate with the proliferative status ofthe kidney cells. To investigate its role in renal epithelial cellulargrowth directly, we studied the human embryonic kidney cell line(HEK-293) stably transfected with either plasmid alone or plasmidcontaining rat HCaRG. [ 3 H]thymidineincorporation was significantly lower in HCaRG clones. Although HCaRG clones exhibited some enhanced susceptibilityto cell death, this was not the primary mechanism of reducedproliferation. Cell cycle analysis revealed a G 2 M phaseaccumulation in HCaRG clones that was associated withupregulation of p21 Cip1/WAF1 and downregulation ofp27 Kip1. HCaRG clones had a greater proteincontent, larger cell size, and released 4.5- to 8-fold more of anatrial natriuretic peptide-like immunoreactivity compared withcontrols. In addition, HCaRG clones demonstrated thepresence of differentiated junctions and a lower incidence of mitoticfigures. Genistein treatment of wild-type HEK-293 cells mimickedseveral phenotypic characteristics associated with HCaRG overexpresssion, including increased cell size and increased release ofatrial natriuretic peptide. Taken together, our results suggest that HCaRG is a regulator of renal epithelial cell growth anddifferentiation causing G 2 M cell cycle arrest.
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novel gene; p21 Cip1/WAF1; p27 Kip1; atrialnatriuretic peptide; human embryonic kidney 293 cells; hypertension-related, calcium-regulated gene
" _( n, g" x" k; n2 ?' v4 e; W 【关键词】 regulator epithelialcell differentiation GMarrest
, h# T3 p- z# }; A2 \ INTRODUCTION
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KIDNEY DEVELOPMENT AND CELLULAR differentiation are dependent on theordered activation of a number of genes whose encoded proteinsdetermine cell phenotype as well as functional responses ( 4 ). The novel hypertension-related, calcium-regulatedgene ( HCaRG ) codes for an intracellular protein, which wehave previously shown (by using in situ hybridization) to be highlyexpressed in the tubular fraction of the kidney and more highlyexpressed in kidneys from hypertensive compared with normotensiveanimals ( 31 ). We have mapped HCaRG to ratchromosome 7 ( 30 ) at a locus determining kidney weight( 14 ). In our previous studies, we also observed that HCaRG mRNA levels declined rapidly in the kidney afterischemia and reperfusion while there was a reciprocal increase in c-myc mRNA ( 31 ). The ischemia-reperfusionmodel of renal injury in vivo is a well-documented stress that resultsin dedifferentiation and mitogenesis of the surviving cells to achievethe repair of the injured kidney ( 10 ). We have also shownthat HCaRG mRNA levels are negatively correlated to theproliferative status of the cell, i.e., they are lower in fetal than inadult kidneys and also downregulated in renal carcinoma. Takentogether, these results suggest that HCaRG could play a rolein the control of renal epithelial cell growth and differentiation forwhich the molecular determinants are still not completely understood.
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& S2 T! x' l1 U, Z0 GAtrial natriuretic peptide (ANP) was originally isolated from mammalianatria and identified as a circulating peptide hormone that is involvedin blood pressure regulation ( 6 ). ANP antagonizes thepressor effects of the renin angiotensin system through its potentnatriuretic, diuretic, and vasorelaxant mechanisms. Extra-atrial expression and synthesis of ANP is now well documented, and previous studies have identified ANP in the brain, ovary, pituitary, lungs ( 12 ), and kidneys ( 20 ) of several species invivo as well as the secretion of ANP by primary cultures of neonataland adult kidney cells ( 26 ). The developmental pattern ofANP immunoreactivity in the rat was shown to coincide with thedifferentiation and maturation of the tubular epithelium( 20 ). The site of local renal ANP synthesis has beenlocalized to the distal tubular epithelial cells of the kidney( 25 ), and studies have identified the local ANPproduced in the kidney as urodilatin, a 32-amino acid peptide consisting of the sequence 95-126 of the ANP prohormone( 28 ). The human embryonic kidney cell line (HEK-293)represents an appropriate dedifferentiated embryonic renal cell line ofhuman origin that exhibits several phenotypic characteristics of distaltubular cells, including the basal synthesis and release of an ANP-like immunoreactivity (or urodilatin) ( 2, 16 ). In the present study, we report that the novel gene HCaRG is a regulator ofHEK-293 cell proliferation, cell cycle progression, and celldifferentiation involving the induction of p21 Cip1/WAF1 andthe downregulation of p27 Kip1.4 ]/ d- z% |+ [7 x" m! P5 F
5 @* d3 v( T3 L3 d9 v5 t- i2 dMATERIALS AND METHODS( L; F% f- T7 j4 S( {, _+ f; w
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Stable transfection and cell culture. HEK-293 cells were transfected with the control plasmid(pcDNA1/ Neo; Invitrogen) or with plasmid containing rat HCaRG (pcDNA1/ Neo - HCaRG ) by using astandard calcium phosphate coprecipitation method and then selected in400 µg/ml G418, and rat HCaRG mRNA levels were determinedas described in detail in previous studies by our laboratory ( 31 ). Clones expressing the highest levels of HCaRG, HCaRG clones 8 and 9, were used in thesestudies, and clones transfected with vector alone, Neo clones 1 and 6, served as controls. Stable transfectants were used atpassage numbers glucose)containing 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mlpenicillin, and 100 µg/ml streptomycin with 400 µg/ml G418.Wild-type HEK-293 cells were maintained in identical conditions butwithout the selection antibiotic G418.: q0 b: h* g& }" d. }1 k/ N
9 q; j7 t/ g1 }Proliferation and apoptosis. Stably transfected or wild-type HEK-293 cells were inoculated in DMEMcontaining 10% FBS in poly- D -lysine-coated plates and thensynchronized for 48 h in DMEM containing 0.2% FBS. This protocol and all subsequent protocols that involved cell synchronization wereconducted for 48 h in DMEM containing 0.2% FBS. Stabletransfectants were then grown for 48 h in DMEM 10% FBS, and 1 µCi [ 3 H]thymidine (ICN) was added for the last 4 hof incubation, as detailed previously ( 31 ). Aftersynchronization, wild-type cells were treated with genistein(1-100 µM) for 48 h and [ 3 H]thymidine wasadded as above ( 31 ). Proliferation was also assessed bycounting cell numbers with a haemocytometer. We used the chromatincleavage assay ( 3 ) to measure apoptosis in stable transfectants growing in DMEM 10% FBS or after serum deprivation (0鸖) as well as under identical conditions to those described for[ 3 H]thymidine incorporation.; B; \: S: J+ X# Q- l
. e5 \, j7 N: \' D* l2 j" qCell cycle analysis. Stably transfected cells were synchronized and then grown in DMEM 10鸖, and a time course was conducted by harvesting cells at 0, 12, 16, 20, 24, and 48 h for cell cycle analysis. Nonsynchronized clonesgrowing in DMEM 10% FBS were also harvested at several time points.Growth curves were constructed for each clone from which the specificgrowth rate was determined by using the formula (P 2 -P 1 )/P 1 ( t 2 - t 1 ),in which P 1 and P 2 are cell density at times t 1 and t 2 as described ina previous study ( 13 ). Wild-type HEK-293 cells weresynchronized and then treated with 50 µM genistein or vehicle, andthe cell cycle was analyzed at 24, 48, and 72 h. Cells wereprepared for flow cytometry by using the CycleTEST PLUS DNA Reagent Kit(Becton-Dickinson) and analyzed with a FACSCalibur benchtop analyzer(Becton-Dickinson). Acquisition of cell cycle histograms was carriedout by using the CellQuest software package with analysis conductedusing the Mod Fit LTV2.0 program.
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6 N( v; |& K$ O7 @Macroarray analysis and Western blotting. HCaRG and Neo clones were synchronized, thengrown in the presence of 10% FBS for 48 h, and then RNA wasextracted by using the RNeasy kit (QIAGEN), treated with DNAse I,purified, and used for preparation of cDNA. Two identical Atlas HumanCell Cycle Arrays (Clontech) were hybridized with human 32 P-labeled HCaRG 8 or HCaRG 9, or 32 P-labeled Neo 1 or Neo 6 cDNAs asprobes, according to the manufacturer's instructions. For Westernblotting, cells were lysed on ice with lysis buffer (10 mMTris · HCl, pH 7.6, 150 mM NaCl, 1% Triton X-100, and 0.1% SDS plus protease inhibitors and EDTA). Cell extracts were mixed with an equal volume of sample buffer, separated by SDS gelelectrophoresis, and then transferred to a polyvinylidene difluoridetransfer membrane (or nitrocellulose membrane for HCaRG Western blot; Amersham Pharmacia Biotech) and incubated with antibodies to p21 Cip1/WAF1 (1:500), p27 Kip1 (1:2,500;Transduction Laboratories), p53 (1:2,500; Calbiochem), and HCaRG (1:500) ( 31 ), followed by horseradishperoxidase-conjugated anti-mouse, anti-goat, or anti-rabbitantibodies. Bands were visualized by using an enhancedchemiluminescence kit, and signals were scanned by densitometry andnormalized to -actin.
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Cell size, protein content, and cell volume. Stably transfected cells were synchronized and then grown in 10% FBS,and protein concentration was measured after 12, 24, 36, 48, and72 h with the modified Lowry method ( 18 ). Cell size was measured by using the National Institutes of Health Image program( http://rsb.info.nih.gov/nih-image/ ) to measure the cross-sectional area of cells at differing degrees of confluency, and the mean cellsize was determined for 100 cells. Cell volume was measured directly byusing the equilibrium distribution of [ 14 C]urea tomeasure the intracellular volume of water per cell as describedpreviously ( 22 ). Wild-type HEK-293 cells were synchronized and then treated with 50 µM genistein or vehicle for 24, 48, or 72 h, and the mean size of 100 cells each was also calculated.. u+ O% g C2 ~4 a, N1 X
5 @% W1 ~7 e3 ~* z' v+ u& c" XMeasurement of ANP. The level of ANP immunoreactivity released into the cell culture mediumwas measured as detailed previously ( 11 ). Samples wereharvested into chilled tubes containing a protease inhibitor solutioncomposed of 10 5 M EDTA, 10 5 M PMSF, and 5 µM pepstatin A and were stored at 80°C. For measurements ofintracellular ANP, cells were pelleted and then resuspended in 1 ml ofPBS containing inhibitor solution, and the intracellular proteins werereleased by conducting three freeze-thaw cycles. Appropriate buffer andincubation media were collected simultaneously and served as thecontrol samples in each ANP radioimmunoassay., z! ]- u" n" ^' |8 }# C' b
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Electron microscopy. Sample preparation for electron microscopy (EM) was carried out asdescribed previously ( 29 ) with some modifications, and then semi-thin sections were stained with toluidine blue and an indexof mitotic figures was obtained by counting cells with condensed chromosomes in 10-15 high-power fields each for confluent cells. From suitable areas, thin sections were cut, double stained on coppergrids with uranyl acetate and lead citrate, and then examined with aPhillips EM 208 transmission electron microscope.4 U/ o9 P/ g* C( L( R
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HCaRG mRNA expression. Wild-type HEK-293 cells were synchronized and then treated with 50 µMgenistein or vehicle for 24, 48, or 72 h. Total RNA was extractedby using the guanidinium isothiocyanate extraction technique, and HCaRG mRNA levels were determined by Northern and Dot blothybridization using a human HCaRG probe with a correction made for GAPDH or poly-A RNA as described previously ( 31 ).
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Statistical analysis. All experiments were conducted two to six times. Results are presentedas means ± SE. Statistical analysis was performed using aStudent's unpaired t -test. P significant.
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. Q5 ?% M& b; S# e, MHCaRG-induced growth suppression is associated with changes in cellmorphology. Wild-type HEK-293 cells that normally express low levels of HCaRG were stably transfected, and HCaRG clones 8 and 9, expressing moderate to high levels of HCaRG, showed areduced proliferation rate and lower cell density at confluencecompared with Neo control clones ( 31 ). Wereport here that the slow-growing HCaRG -transfected cells showed marked changes in morphology and size comparedwith the control cells (Fig. 1 A ). The Neo control clones, which exhibit a small, polygonal-shaped,epithelial-like morphology, continued to grow exponentially and packedtogether closely at confluence (see A and C in Fig. 1 A ),which was the growth pattern we observed for wild-type cells (notshown). However, as a result of HCaRG overexpression, theHEK-293 cells exhibited a larger surface area with the presence of verylarge, flattened cells (and/or cytoplasmic vacuoles; see B in Fig. 1 A ) and the cells did not pack together as closely atconfluency (see D in Fig. 1 A ), consistent with reaching alower saturation density. The overexpression of the HCaRG protein in HCaRG clones 8 and 9 was confirmed with Westernblot analysis (Fig. 1 B ). We assessed the effect of HCaRG overexpression on cell death and found some enhancedsusceptibility to apoptosis in response to complete serumdeprivation ( Neo, 5.5 ± 0.3%, vs. HCaRG,11.2 ± 0.8%; n = 3; P in the level of apoptosis between Neo and HCaRG clones after synchronization in 0.2% serum for48 h ( Neo, 3.13 ± 0.33%, vs. HCaRG,3.55 ± 0.18%; n = 8; not significant) or whenreturned to 10% serum for an additional 48 h ( Neo,2.73 ± 0.6%, vs. HCaRG, 4.6 ± 0.34%; n = 4; not significant), thus confirming the minimalcontribution of apoptosis to the observed reduction inproliferation as observed using [ 3 H]thymidineincorporation.* k. c, L k' Z: X
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Fig. 1. HCaRG -overexpressing cells exhibit changes inmorphology. A : HEK-293 cells stably transfected with eitherplasmid alone ( Neo clone 1; A and C) or plasmid containingrat HCaRG ( HCaRG clone 9; B and D) werephotographed at low and high density in normal growth conditions.Photographs illustrate the difference in cell size and morphology (Aand B) as well as the difference in cell density attained at confluency(C and D). We noted very large flattened cells and/or cytoplasmicvacuoles that were evident in HCaRG -transfected cell linesonly (arrows in B and D). B : representative Western blotshowing overexpression of HCaRG protein in HCaRG clones 8 and 9 compared with Neo control clones 1 and 6. HCaRG protein and molecular weight standard are asindicated. NS, nonspecific binding.
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Cell cycle progression is blocked at G 2 M phase inHCaRG-overexpressing clones. Representative cell cycle DNA histograms for nonsynchronized activelygrowing cells are shown in Fig. 2, A and B, and the HCaRG cells show anaccumulation in G 2 M phase, suggesting a putative G 2 M arrest. A detailed assessment of cell cycle progressionafter addition of serum to synchronized cells was conducted thatclearly showed a significant increase in G 2 M phase( Neo vs. HCaRG; n = 6 at 24 and48 h; P G 1 phase in HCaRG cells (Fig. 2, E and C ). Specific growth rates for each cloneunder normal growth conditions showed the greatest difference between days 2 and 4 after plating (average values for Neo cells, 1.32 ± 0.02, compared with HCaRG cells, 0.85 ± 0.06; 1.6-fold difference). Cell cycle distributionin each clone after 2 and 4 days of growth was analyzed and confirmedthe G 1 phase reduction ( Neo vs. HCaRG; n = 4 for days 2 and 4; P and also confirmed thatG 2 M phase accumulation ( Neo vs. HCaRG; n = 4 for days 2 and 4; P between the Neo and HCaRG cell lines(Fig. 2 F ).
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$ Z, e( H, V4 M0 C5 b0 { VFig. 2. The effect of HCaRG overexpression on cellcycle progression. Representative cell cycle histograms fornonsynchronized Neo control cells ( A ) and HCaRG cells ( B ) after 48 h growth areindicated. C-E : time course of cell cycle progressionafter synchronization. F : cell cycle distribution innonsynchronized cells after 2 and 4 days of growth. Values aremeans ± SE of n = 2-4 each for Neo and HCaRG at each time point.5 R j- c+ J6 e5 H4 _
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HCaRG-induced growth suppression is associated with increasedp21 Cip1/WAF1 and decreased p27 Kip1. We compared the differential gene expression profile of 111 cell cycleregulatory genes in the stably transfected clones by using a humanAtlas macroarray. Macroarray analysis was conducted in two HCaRG clones ( HCaRG 8 and HCaRG 9) andin two Neo control clones ( Neo 1 and Neo 6). Representative filters for Neo 1 and HCaRG 9 are presented in Fig. 3, A and B. Thearrows indicate a small subset of upregulated genes in HCaRG cells. The results for differential expression in HCaRG clones 8 and 9 compared with Neo clones 1 and 6 aresummarized in Fig. 3 C. The gene whose expression wasmaximally induced in both HCaRG clones was thecyclin-dependent kinase (CDK) inhibitor p21 Cip1/WAF1 (average 2.5-fold upregulation). Other genes that were upregulated include cyclin D1 (average 1.7-fold); gadd153 (average 1.8-fold); CDKL1(average 1.9-fold); Jun-B (average 2-fold), and Big MAPK-1/ERK5 (average 1.6-fold) upregulation. Western blot analysis showed upregulation of p21 Cip1/WAF1 protein levels in HCaRG clones 8 and 9 ( Neo vs. HCaRG, n = 6; P 3 D ). Protein levels of theG 1 /S-specific CDK inhibitor p27 Kip1 weredownregulated in HCaRG clones 8 and 9 ( Neo vs. HCaRG, n = 4; P 3 E ), whereas there was no difference in p53 levels asnoted by both macroarray analysis and Western blotting (not shown).
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8 z6 T) C9 m5 Z5 nFig. 3. The effect of HCaRG overexpression on cell cycle geneexpression. A and B : representative filters for Neo 1 and HCaRG 9. Arrows, small subset ofupregulated genes. C : macroarray analysis of differentiallyexpressed genes with data presented as fold upregulation in HCaRG clones 8 and 9 compared with Neo clones 1 and 6. AC #; accession number. Immunoblotting results from synchronizedcells after 48 h growth show that HCaRG clones 8 and 9 have increased p21 Cip1/WAF1 ( D ) and decreasedp27 Kip1 ( E ) protein levels. Values aremeans ± SE.
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& Q0 N, G! p! Y m2 J6 eHCaRG-induced growth suppression is associated with cellularhypertrophy and increased levels of ANP. HCaRG -transfected cells had a significantly greater totalprotein content per cell when compared with Neo controlcells at several time points, and Fig. 4 A presents the results after48 h growth ( Neo vs. HCaRG, n = 4-6; P HCaRG may be causing cellular hypertrophy. This featurewas confirmed by direct measurement of cell size, which showed asignificant increase in HCaRG cell size compared with Neo control cell size ( Neo 1, Neo 6 vs. HCaRG 8, HCaRG 9, n = 100 cells each clone; P 4 B ).Furthermore, the volume of the HCaRG -transfected cellsmeasured as [ 14 C]urea-available space was significantlygreater compared with the Neo control cells ( Neo vs. HCaRG, n = 6; P 4 C ). HCaRG cells showed asignificant increase both in the intracellular content ( Neo vs. HCaRG, n = 4; P 4 D ) and in the release of ANP ( Neo vs. HCaRG, n = 6; P 4 E ). However, there was no significant difference inthe ratios of secreted to cellular ANP between the control and the HCaRG -transfected cell lines (extracellular ANP/intracellular ANP ratios for each clone were as follows: Neo 1, 0.04; Neo 6, 0.07; HCaRG 8, 0.03; and HCaRG 9, 0.06). This suggests that HCaRG overexpression in HEK-293 cells causes increased synthesis and release of ANP in a constitutive manner.3 n1 _4 i* n( ^0 k" f6 p6 v
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Fig. 4. The effect of HCaRG overexpression on cellprotein content, cell size, cell volume, and atrial natriureticpeptide. A : HCaRG -transfected cells had anincreased total protein content per cell compared with Neo control cells ( n = 3 for each clone); representativeexperiment of 2 with this protocol. B : cell size analysisshowed that HCaRG cells are larger than Neo control cells ( n = 100 for each clone). C : HCaRG -transfected cells had an increased volume ofintracellular water compared with Neo control cells( n = 3 for each clone). HCaRG -transfectedcells synthesized ( D ) and released ( E ) more of anatrial natriuretic peptide (ANP)-like immunoreactivity compared withcontrols. Representative (longer term) experiment of 2 with thisprotocol in duplicate ( D ) or triplicate ( E ).Values are means ± SE.
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2 v' p V3 C2 p& S% jFeatures of cellular differentiation identified with EM. Sections of fixed cell preparations from confluent (Fig. 5, A and B ) andsubconfluent (Fig. 5, C and D ) cell lines wereexamined by a pathologist who first confirmed the epithelial phenotype of the cells. There was a lower incidence of mitotic figures in the HCaRG -transfected cells compared with the Neo control cells ( Neo, 13.6 ± 1.0, vs. HCaRG,6.3 ± 0.3; n = 10-15 high-power fields; P 2 phase. Transmission EM ultrastructural analysis revealedthe presence of more differentiated junctions (desmosome-likejunctions) in the HCaRG cells only (Fig. 5 B ),with the Neo control cells showing the presence of tight junctions (Fig. 5 A ). In addition, HCaRG cellsdemonstrated features consistent with junctional (glandular-like)complex formation and numerous microvilli (Fig. 5, C and D ).
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2 P: M3 D6 U( Z( b: n. ~6 G+ OFig. 5. Electron microscopy of HCaRG stably transfectedHEK-293 cells. HCaRG -transfected cells havedesmosome-like junctions ( B ), which were not present in Neo control cells ( A ). Magnification,×40,000. C : HCaRG -transfected cells havenumerous microvilli (M) and also features of junctional complex(JC) formation ( inset ). Magnification, ×33,500. D : higher magnification (×50,000) image of the C, inset, depicts features consistent withjunctional complex formation., U) u1 U/ R+ T
$ h3 v* n2 I1 h S6 X! L# s* WTreatment of wild-type HEK-293 cells with genistein. Because our data suggested that HCaRG was causing growthsuppression in association with the induction of a more differentiated phenotype, G 2 M arrest and altered cell size, we conductedthe converse experiment in which we treated the wild-type HEK-293 cellswith an agent, namely, genistein, that is known to act at theG 2 M phase to alter the differentiation status of the cells ( 19, 21, 24 ). We found that treating HEK-293 cells with 50 µM genistein for 48 h resulted in a similar decrease inproliferation (Fig. 6 A ) andincrease in cell size (Fig. 6 B ) as was documented in HCaRG -transfected cells. The G 2 M arrest ingenistein-treated cells was more marked than that observed with HCaRG overexpression, but in both models there was nopolyploid population observed, as assessed by flow cytometry cell cycleDNA histogram analysis. HCaRG overexpression causedupregulation of p21 Cip1/WAF1, but genistein treatment didnot cause any change in p21 Cip1/WAF1 levels (not shown).Genistein treatment for 48 h caused a 1.9-fold increase in thelevel of ANP release per cell (Fig. 6 C ), which was less thanthat observed in response to HCaRG overexpression, whichcaused a 4.5-fold increase after 48 h. Concomitant with theincrease in ANP release, wild-type HEK-293 cells exposed to genisteinexhibited an increase in endogenous HCaRG mRNA levels compared with control cells (Fig. 6 D ).9 s3 c8 X* ?& G9 S( s
; \/ |' m2 O# ~; F IFig. 6. Effects of genistein treatment on wild-type HEK-293 cells. A : cell proliferation ( n = 4). B :cell size ( n = 100) in wild-type HEK-293 control cellsand in genistein-treated cells (50 µM, 48 h). C :atrial natriuretic peptide release ( n = 4). D : HCaRG mRNA levels ( n = 2-4) in wild-type HEK-293 control cells and in genistein-treatedcells (50 µM, 48 h). Values are means ± SE.
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5 L/ c8 ]- H+ w' ?, b3 }DISCUSSION
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9 G& r0 W) N, h0 S- t& {1 cIn our previous studies, we have shown that HCaRG mRNAlevels are negatively correlated with the proliferative cell status inthe kidney in vivo ( 31 ). The purpose of this study was to investigate the cellular function and mechanisms whereby the novel gene HCaRG affects cell proliferation. To address this issue, wehave characterized the effects of HCaRG overexpression inHEK-293 cells, a cell line that retains several characteristics ofrenal epithelial cells. We report here that the slow growth of the HCaRG clones is associated with a noticeable change in cellmorphology and increased cell size, suggesting a change in thedifferentiation status of the embryonic kidney cells. We conducted cellcycle analysis by using synchronized and nonsynchronized cells andfound that HCaRG cells accumulated at the G 2 Mphase in both conditions. Because the specific growth rate was lower in HCaRG clones compared with controls mainly between day2 and day 4 of growth, we analyzed the cell cycledistribution at these time points, which confirmed a G 2 Marrest. Cell cycle arrest in the G 2 M phase is frequently found to be associated with DNA endoreplication and hypertrophy ( 7 ). However, we found no evidence of polyploidy inresponse to HCaRG overexpression in the HEK-293 cells. It isrelevant that our analysis of the HCaRG promoter hasdocumented the presence of CHR (cell cycle gene homology region) andCDE (cell cycle-dependent element) regulatory elements (Tremblay S andTremblay J, unpublished observations), which suggests that HCaRG may be expressed in a cell cycle-dependentfashion at the G 2 M phase ( 34 ).
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! j9 k6 Q2 e5 ~! jCell cycle-related growth suppression is frequently mediated by theinduction of the CDK inhibitor p21 Cip1/WAF1, which canact to cause arrest at the G 1 or G 2 M phase ofthe cell cycle by p53-dependent or p53-independent means ( 23, 32 ). Although hypertrophy of renal epithelial cells is known tobe associated with G 1 arrest due to upregulation ofp27 Kip1, a previous report has shown growthsuppression in HEK-293 cells due to G 2 M phase arrestand sustained induction of p21 Cip1/WAF1 ( 9 ).To investigate the mechanisms of HCaRG -induced cell cycle arrest, we conducted macroarray differential gene expression analysis in the stably transfected clones. The gene whose expression was upregulated the most in both HCaRG clones wasp21 Cip1/WAF1, which is known to be increased in associationwith differentiation induction. Other genes that were upregulated inboth clones were cyclin D1 and gadd153, known tobe involved in growth arrest in association with pathways ofdifferentiation and/or apoptosis ( 33 ). Jun-B isknown to be upregulated during cell growth suppression anddifferentiation in embryonic tissues ( 27 ), suggesting a maturation of the embryonic kidney cells in response to HCaRG overexpression. The redox-sensitive Big MAPK-1/ERK5 isupregulated in response to H 2 O 2 and osmoticstress and has also been linked to pathways of cellular differentiation( 8 ).
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* ~( w5 e# @. n6 r2 r% ZBecause of the observed upregulation of p21 Cip1/WAF1 mRNA,we conducted Western blotting, which confirmed thatp21 Cip1/WAF1 was also upregulated at the protein level inboth HCaRG clones. Because of the role ofp27 Kip1 on G 1 /S phase progression, we conductedWestern blotting, which showed that p27 Kip1 wasdownregulated in both HCaRG clones, whereas macroarray and Western blot analyses showed no change in p53 levels.Therefore, elevated p21 Cip1/WAF1 levels along withincreased cyclin D1 expression and downregulated p27 Kip1 isconsistent with a successful G 1 /S phase transition, withgrowth arrest occurring at the G 2 M phase. The reciprocalrelationship between the CDK inhibitors p21 Cip1/WAF1 andp27 Kip1 is interesting and possibly changes in thatstoichiometry could play a pivotal role in the HCaRG -associated G 2 M phase arrest." i% L) n! g0 A9 X) ?# Y# v q
( Z& F8 O. z+ K" s l* ]The HEK-293 cells release a basal level of ANP-like immunoreactivity( 2, 16 ). In the present study, HCaRG overexpression caused a 4.5- to 8-fold increase in ANP levels andincreased size and protein content. The increased levels of ANP may bea consequence of the induction of differentiation per se or,alternatively, may be a marker of hypertrophy, as is well known tooccur in myocytes ( 5 ). The distal tubular cells of thekidney are known to produce ANP locally for subsequent release to actdownstream at the inner medullary collecting duct as part of aparacrine short feedback loop ( 20 ). The upregulation ofANP that we observe in HEK-293 cells in response to HCaRG overexpression in vitro may reflect such a paracrine functionalresponse in keeping with this in vivo role of the distal tubularepithelial cells.2 z8 E$ u9 F0 [3 e$ C- e
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Previous studies in vascular smooth muscle cells have demonstrated therole of p27 Kip1 in mediating ANG II-induced hypertrophy( 1 ). ANP has been shown to antagonize ANG II-induced renaltubular cell hypertrophy (at the G 1 /S phase) throughmechanisms involving the downregulation of p27 Kip1 ( 15 ). It is possible to hypothesize that the elevated ANPrelease in HEK-293 cells due to HCaRG overexpression exertsan autocrine action to offset G 1 /S arrest by downregulatingp27 Kip1 levels. In vivo, it is known that local renal ANPlevels are modulated in the kidney in response to changes in the localrenin angiotensin system in a blood pressure-independent manner. In themodel of renal compensatory hypertrophy, increased local renal ANP wassuggested to maintain renal function and attenuate the hypertrophicgrowth response ( 17 ).
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! q7 P% N& E5 I8 x; @Genistein is known to reduce cell growth in association withdifferentiation in several cell lines, including those derived from thekidney ( 21 ). For this reason, we treated wild-type HEK-293cells with genistein, which mimicked several of the phenotypes associated with HCaRG overexpression, including reduced cellgrowth, G 2 M arrest, increased cell size, and increased ANPrelease. Although treatment with genistein did not induceupregulation of ANP to the same extent as HCaRG overexpression, the increase was concomitant with a similar foldincrease in endogenous HCaRG expression, suggesting apossible association of HCaRG with hypertrophy and/ordifferentiation. However, the changes in HCaRG mRNA levelsobserved may equally be a secondary effect of the growth retardationcaused by genistein.& x' A2 L& K( K
5 o2 H( u9 T% x' i- `7 |: m( r5 PWe also conducted EM analysis of the HCaRG cells, whichrevealed features of epithelial differentiation at the cellultrastructural level, including the presence of desmosomes as well asmicrovilli and other features consistent with junctional complexformation. The mitotic index was significantly reduced in HCaRG cells, which is indicative of a block in the cellcycle at the G 2 phase. Taken together, these observationsprovide additional information that suggests that HCaRG isassociated with cell growth suppression and the induction of a moredifferentiated epithelial cell structure.
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5 W+ _! Y% z( r. O9 u( E! I) XIn the present studies, we report that HCaRG -induced growthsuppression is associated with reduced specific growth rate and mitoticindex, cell cycle G 2 M arrest, increased levels ofp21 Cip1/WAF1, and decreased levels of p27 Kip1,as well as a change in cell phenotype involving altered cell morphology, increased cell size and cell volume, increased synthesis and release of ANP, and the presence of desmosomes. Suchphenotypic changes in response to HCaRG overexpression areindicative of a cellular differentiation program, and we have alsoidentified a new component of cell cycle progression involving HCaRG. However, it is pertinent to note that the HEK-293cell line is transformed and as such may exhibit different growthresponses compared with a nontransformed cell type. Despite thislimitation, it is reasonable to speculate on the basis of the presentstudies that HCaRG may represent a novel target forintervention in proliferative disorders.. ~! o5 m! f$ t, U; i3 C
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ACKNOWLEDGEMENTS0 F9 E6 Q( |1 N) h$ k
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We thank Myrielle Vermette (Dept. of Pathology, Centre deRecherche, Centre Hospitalier de l'Université de Montréal)for help with the EM analysis.
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