Nestin is an intermediate filament protein originally described in neural stem cells and a variety of progenitor cells. More recently, nestin was detected in rat kidney podocytes. We show here that nestin is expressed in a developmentally regulated pattern in the kidney. Nestin was detected by immunohistochemistry in the condensing mesenchyme surrounding the ureter, in developing glomeruli, in podocytes of the adult kidney, and in a podocyte cell line. Nestin shared a striking overlap in expression with the Wilms' tumor suppressor Wt1. Nestin was significantly upregulated in a cell line with inducible Wt1 expression upon induction of Wt1. Cotransfection experiments in human embryonic kidney cells (HEK293) revealed stimulation of a nestin intron 2 enhancer element up to six-fold by the Wt1(-KTS) splice variant. Nestin expression was significantly reduced in an inducible mouse model of glomerular disease. This model is based on podocyte-specific overexpression of Pax2 and associated with a loss of Wt1 expression (33). Furthermore, also in the developing heart, nestin was found in an overlapping pattern with Wt1 in the epicardium and the forming coronary vessels. Strikingly, in the hearts of Wt1 knockout mice, nestin was barely detectable compared with the hearts of wild-type embryos. Our results show that nestin is expressed at different stages of kidney and cardiac development and suggest that its expression in these organs might be regulated by the Wilms' tumor suppressor Wt1.
- glomerular disease
- coronary vessel
- intronic enhancer
nestin is an intermediate filament protein, which was originally identified in neuroepithelial stem cells of rat, mouse, and human (20, 6, 40). Nestin shows structural similarities to type III and type IV intermediate filament proteins, but because of the relatively low degree of protein sequence homology to the known five classes of intermediate filament proteins, nestin was defined as a new class VI intermediate filament (21). Because of a very short N-terminus, nestin is unable to self-assemble and thus, requires interaction with other intermediate filament proteins like desmin, vimentin, or glial acidic protein (for a review, see Ref. 39). Besides the unusual structural features, also regulation of nestin seems to be unique. Analyses of the rat promoter in transgenic mice indicate that nestin expression is regulated via enhancer elements in the first and second intron rather than the 5′-upstream region (41). It has been suggested that enhancer elements in the first intron stimulate nestin expression in angiogenic endothelium (1), whereas enhancer elements in the second intron are required for expression in neuronal progenitor cells (41). As transcriptional regulators of nestin Pou, Sox (14, 31), and the thyroid transcription factor-1 (22) have been identified until now.
Nestin has been considered as a marker for stem cells and progenitor cells in a variety of organs; that is, it was detected in neuronal precursor cells, developing skeletal and cardiac muscle cells, mesonephric mesenchyme, pancreatic progenitor cells, and vascular endothelium among others (for a review, see Ref. 39). It has been thought that nestin expression is transient in these progenitor cells, and differentiation in specialized cells types results in downregulation of nestin and expression of other cell type-specific intermediate filaments, for example, neurofilaments in neurons (5). Nestin expression in adult tissues was attributed to stem cell and progenitor cell populations, and it was postulated that these progenitor cells are reactivated and proliferate and migrate in response to injury during tissue regeneration (for a review, see Ref. 39). A recent study showing nestin expression in newly forming blood vessels and a high number of nestin-positive endothelial cells after myocardial infarction might be in contrast to the concept of nestin expression exclusively in progenitor cell populations (24). Another group reported nestin expression in rat kidney podocytes (42), which is again in conflict with nestin expression exclusively in proliferating progenitor cells, as podocytes are regarded as terminally differentiated postmitotic cells.
Thus we analyzed whether nestin might be expressed in cell types independent of progenitor populations and describe nestin expression in the developing kidney and heart. Furthermore, we investigated molecular mechanisms, which might contribute to nestin expression in these tissues and establish a role for the Wilms' tumor suppressor Wt1 in the regulation of nestin expression.
MATERIALS AND METHODS
All protocols used were performed in accordance with the National Institutes of Health “Guidelines for the Care and Use of Laboratory Animals” and were approved by the Institutional Animal Care and Use Committee of the University of Nice. A heterozygous (Wt1+/−) breeding pair [C57BL/6 strain] was obtained from the Jackson Laboratory (Bar Harbor, ME) and genotyped by PCR according to the protocol provided. Inducible double transgenic Z/Pax2;CreTx mice were generated by crossing Z/Pax2 transgenic animals with tamoxifen-inducible Cre-deleter mice (4, 33). Double transgenic offspring was identified by PCR using the following primers: YAC-F 5′-ACTTCACTCGGGCCTTGATAG-3′ (forward primer); YAC-B 5′-GTGGAGAGTCAGACTTGAAAG-3′ (reverse primer); Cre-F 5′-CGCAGAACCTGAAGATGTTCGCGA-3′ (forward primer); Cre-B 5′-GGATCATCAGCTACACCAGAGACG-3′ (reverse primer). Tamoxifen induction was performed as described (4, 33).
Immunohistochemistry and immunocytochemistry.
Staged embryos (the morning of vaginal plug was considered E0.5) and isolated organs from adult mice were fixed overnight at 4°C in 3% paraformaldehyde in PBS and embedded in paraffin. Three-micrometer paraffin sections were cut and transferred onto gelatin-coated glass slides. Tissue sections were permeabilized with 0.1% Triton X-100 in PBS and blocked by incubation for 1 h in 10% normal serum (in PBS, 0.1% Triton X-100, 3% BSA), which was obtained from the same species as the secondary antibody. Sections were incubated (16 h, 4°C) with primary antibodies diluted 1:100 in PBS, 0.1% Triton X-100, 3% BSA: anti-Nestin antibody from mouse (MAB353, Chemicon, Temecula, CA), anti-Wt1 antibody from rabbit (C-19, sc-846, Santa Cruz Biotechnology, Santa Cruz, CA). Tissue sections were stained with a peroxidase technique. For the mouse monoclonal antibody, the antigen detection was performed with Vector M.O.M. immunodetection Kit (Vector Laboratories, PK-2200, Burlingame, CA), for the polyclonal antibody with a biotinylated antibody against rabbit (Vector Laboratories), followed by incubation with peroxidase coupled streptavidin (Sigma, St. Louis, MO). Visualization was achieved with diaminobenzidine substrate (Vector Laboratories, cat. # SK-4100); in the case of the double-labeling of Wt1 and Nestin, the latter was visualized using VIP substrate (Vector Laboratories, cat. # SK-4600). An indirect immunofluorescence double-labeling technique was used to mark Wt1 and nestin expressing cells (32). In case of cultured podocytes, these were after washing with PBS fixed with 3% paraformaldehyde. The podocytes or sections were incubated (16 h, 4°C) with primary antibodies each diluted 1:100 in PBS, 0.1% Triton X-100, 3% BSA: polyclonal anti-Wt1 from rabbit (C-19, sc-846, Santa Cruz Biotechnology) and monoclonal anti-nestin from mouse (MAB353, Chemicon). The reaction products were visualized by incubation (1.5 h, room temperature) with Cy2- and Cy3-conjugated secondary antibodies. Slides were viewed under an epifluorescence microscope (DMLB, Leica, Wetzlar, Germany) connected to a digital camera (Spot RT Slider, Diagnostic Instruments, Livingston, Scotland) with the Spot software (Universal Imaging, Downingtown, PA) or alternatively with a Zeiss 2 photon confocal microscope.
Nestin reporter constructs.
Sequences of the human nestin gene (NCBI accession no. AF004335) were amplified from blood cell-derived DNA using the Expand Long Template PCR System (Roche Diagnostics, Mannheim, Germany). Sequence information of the primers that were used for PCR amplification of intron 2 sequence of the nestin gene is given in Table 1. The PCR products were ligated into the KpnI and HindIII sites of the pGL2basic reporter plasmid (Promega) and verified by automated dideoxy sequencing.
Human embryonic kidney (HEK) 293 cells (accession no. ACC 305) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Cells were grown in DMEM (PAA Laboratories, Pasching, Austria) supplemented with 10% FCS (Biochrom KG, Berlin, Germany), 100 IU/ml penicillin (Invitrogen, Karlsruhe, Germany), and 100 μg/ml streptomycin (Invitrogen) and routinely split at a 1:10 ratio twice per week. Mouse podocytes were a gift of K. Endlich (29). The podocytes carrying a temperature-sensitive mutant of the immortalizing SV40 large T antigen under control of the interferon-γ-inducible H-2Kb promoter (13, 26) were kept in RPMI 1640 nutrient (Invitrogen), supplemented with 10% FCS (Invitrogen), 100 IU/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen) and were cultivated at 32°C in the presence of 10 IU/ml mouse recombinant interferon-γ (Invitrogen, permissive conditions) or at 38°C without interferon-γ for a minimum of 2 wk to induce differentiation (29). Stable U2OS cells (clone UB27) with the tetracycline repressible Wt1(-KTS) form were grown as described (8).
Cell transfection experiments and reporter gene assays.
For transfection experiments, the HEK293 cells were grown to ∼60% confluence in 24-well tissue culture plates. One hundred nanograms of the reporter constructs together with 25 ng of a cytomegalovirus (CMV)-driven β-galactosidase plasmid and 125 ng of expression constructs encoding different Wt1 forms were transiently cotransfected with the use of the Fugene6 reagent (1 μl per well) according to the supplier's protocol (Roche Diagnostics). The pGl2basic vector (Promega, Mannheim, Germany) and the pCB6+ plasmid were transfected for control purposes. Luciferase activities were measured in the cell lysates after 48 h using the firefly luciferase system (Promega) on a Microlite TLX1 luminometer (Dynatech Laboratory, Alexandria, VA). A Beckman DU604 spectrophotometer (Beckman Coulter, Krefeld, Germany) was used for determination of β-galactosidase activities in each sample (34, 35, 37). Values are presented as relative light units normalized to β-galactosidase activities for internal control of transfection efficiencies. The results shown are averages of at least three transfection experiments each performed in duplicate.
RT-PCR was performed with 2 μg of total RNA, as described elsewhere (37, 38). Real-time PCR was performed on the Light Cycler Instrument (Roche) using the Platinum SYBR Green kit (Invitrogen). The following primers were used for PCR amplification: mouse nestin (NCBI accession no. NM_016701), 5′-CTGCAGGCCACTGAAAAGTT-3′ (forward primer), 5′-GACCCTGCTTCTCCTGCTC-3′ (reverse primer); mouse Wt1 (NCBI accession no. NM_144783), 5′-AGAGCCAGCCTACCATCCGCAA-3′ (forward primer), 5′-GGCTGCCTGTGCAACTGTCA-3′ (reverse primer), mouse GAPDH (NCBI accession no. M32599), 5′-ATTCAACGGCACAGTCAAGG-3′ (forward primer), 5′-TGGATGCAGGGATGATGTTC-3′ (reverse primer); human nestin (NCBI accession no. NM_006617.1), 5′-GGCAGCGTTGGAACAGAG GT-3′ (forward primer), 5′-CATCTTGAGGTGCGCCAGCT-3′ (reverse primer); human GAPDH (NCBI accession no. NM_002046.3), 5′-GAAGGTGAAGGTCGGAGTCA-3′ (forward primer), 5′-CAGGAGGCATTGCTGATGAT-3′ (reverse primer).
ANOVA with Bonferroni test as post hoc test and Student’s t-test were performed as indicated. A P value of less than 0.05 was considered statistically significant.
RESULTS AND DISCUSSION
Nestin is expressed in the developing and adult kidney.
Although nestin expression has been described in a variety of developing organs and cell types, for example, neuronal precursor cells (10), developing cardiomyocytes (15), vascular endothelial cells (24), or Sertoli cells (11); until recently, beside one report on nestin expression in rat podocytes (42), little was known about nestin expression during normal embryonic kidney development. We could show here that nestin is expressed at embryonic day 12.5 (E12.5), the earliest time point studied, in the condensing mesenchyme surrounding the ureter (Fig. 1A), although we cannot exclude that nestin is expressed already earlier. At later stages of development, nestin expression is prominent in the more mature glomeruli in the deep cortex of the kidney, but also detectable in more immature glomerular progenitors, that is, comma- and s-shaped bodies, and in the most outer kidney cortex in mesenchymal progenitor cells surrounding the ureteric bud branches (Fig. 1B). In the adult kidney, nestin expression is restricted to the glomeruli (Fig. 1C), which is in agreement to a previous report on nestin expression in rat podocytes (42).
Nestin and Wt1 are upregulated in podocytes upon induced differentiation.
To analyze the expression and subcellular localization of nestin in more detail, we made use of a recently established mouse podocyte cell line, which carries a temperature-sensitive mutant of the SV40 large T antigen and can be differentiated into a podocyte cell type by culturing at 38°C (29). It has been shown that these differentiated cells express the podocyte proteins nephrin, podocalyxin, podocin, CD2AP, synaptopodin, and Wt1, and they form foot process-like structures (29). Interestingly, in the great majority of differentiated podocytes, we could also detect nestin expression (Fig. 2). Nestin showed the typical filamentous staining in differentiated cells, which are characterized by nuclear Wt1 expression. Furthermore, nestin protein was enriched in the developing processes of the cells, which might resemble foot process-like structures (Fig. 2B). To analyze whether nestin might be differentially expressed in differentiated vs. undifferentiated podocytes, we performed quantitative RT-PCR as described (33, 37) and detected a significant increase in nestin expression upon induced differentiation of the podocytes (Fig. 2C). Interestingly, also the expression of the major podocyte transcription factor Wt1 became upregulated in differentiated vs. undifferentiated cells (Fig. 2D).
Nestin and Wt1 share an overlapping expression pattern in the kidney in vivo.
Because the nestin expression pattern during kidney development closely resembles the pattern described for Wt1 (2, 30) and the expression of both Wt1 and nestin is overlapping in the podocyte cell line and changed in a comparable manner during induced differentiation of these cells, we investigated by immunohistochemical double-staining whether both proteins show an overlapping expression in the kidney in vivo. At embryonic day 12.5, the earliest time point studied, high Wt1 expression was detected in the condensing mesenchyme surrounding the branches of the ureteric bud, whereas Wt1 signal was weak in the loose mesenchyme, as reported (2, 30). The Wt1-positive cells in the condensing mesenchyme showed coexpression of nestin (Fig. 3A). Interestingly, nestin was enriched mostly in the basal parts of the mesenchymal cells, which face the ureter. At embryonic day 15.5, nestin and Wt1 shared an overlapping expression in glomerular precursors (Fig. 3B), where again, nestin showed localization in the basal parts of Wt1-positive cells. Confocal microscopy revealed nuclear Wt1 expression and nestin protein localization in the cytoplasm and processes of podocytes in glomeruli of the adult mouse kidney (Fig. 3C), which is in agreement with the observation of Chen et al. (6). This specialized localization of nestin during kidney development and in podocytes in culture might suggest a role in cell polarity or cell process formation, although future studies are required to support this speculation.
Wt1 stimulates nestin directly.
The colocalization of nestin and Wt1 throughout kidney development raised the interesting possibility that Wt1, which acts as a major transcriptional regulator (for a review, see Ref. 28), might directly stimulate nestin expression. More than 20 different Wt1 proteins are generated by alternative mRNA splicing (12), the use of alternative translation start sites (3, 27), and RNA editing (30). The major isoforms are generated by alternative splicing of exon 5, which encodes 17 amino acids, and the use of two alternative splice donor sites at the end of exon 9, which results in the insertion/omission of a tripeptide (lysine-threonine-serine, KTS) in the zinc finger domain of the Wt1 protein (12). The Wt1(+KTS) forms with the tripeptide insertion have a presumed role in mRNA processing (7, 9, 16, 18), whereas Wt1(-KTS) proteins, which lack the KTS peptide, function mainly as transcription factors (reviewed in Refs. 19 and 28).
To analyze a potential direct stimulation of nestin by Wt1, we made use of a stable osteosarcoma-derived cell line (U2OS cells), which expresses the Wt1(-KTS) splice variant under control of a tetracycline repressed promoter (8). Removal of tetracycline from the culture medium resulted in a nearly sixfold increase in the expression of nestin as determined by quantitative RT-PCR (Fig. 4A), indicating that Wt1 could stimulate expression of nestin. Because it is known that nestin is mainly regulated by stimulation of intronic enhancer elements (1, 14, 22, 23, 31), we addressed the question whether Wt1 would activate an intronic enhancer of the nestin gene directly. For this purpose, a 1.7 kb sequence containing intron 2 from the human nestin gene (NCBI accession no. AF004335), as well as several deletion mutants were cloned and ligated into the pGL2 basic reporter plasmid (Fig. 4B). The luciferase reporter was transiently transfected into HEK293 cells along with Wt1 expression constructs and a CMV-β-gal plasmid for normalization of transfection efficiencies. Cotransfection of Wt1(-KTS) stimulated the activity of the nestin intron 2 enhancer significantly, whereas Wt1(+KTS) had no effect (Fig. 4C). To narrow down the region containing elements for activation of the nestin enhancer by Wt1, we cotransfected several reporter constructs with different 3′- or 5′-truncations in HEK293 cells. The transfection experiments yielded a ∼300-bp sequence that was required for stimulation of the nestin enhancer by Wt1 (Fig. 4B). Constructs, which contained nestin 5′- or 3′-intron 2 sequences of the identified 300-bp regulatory sequence were not stimulated by Wt1 (Fig. 4C,e), indicating specificity of the 300-bp element for activation by Wt1. Also, a reporter construct containing 2.9 kb of 5′ upstream sequence of the nestin gene was stimulated neither by Wt1(-KTS) nor by Wt1(+KTS) (0.99 ± 0.09 and 0.95 ± 0.1, respectively). Sequence analysis of the identified 300-bp intron sequence that was required for stimulation by Wt1 revealed a high GC content of the sequence, which is typical for Wt1 binding sites (8). We could detect a potential binding element (5′- GGGAGGACGTGGAGGAGAGGGG-3′), which showed high similarity to the Wt1 binding site in the nephrin promoter (38), although we did not confirm binding to Wt1 protein experimentally.
Besides the activation of nestin by Wt1, which we showed here, a variety of transcription factors, that is, Sox1/2/3, Sox11 (31), Brn-1, Brn-2, Brn-4 (14), and TTF-1 (22) have been reported to stimulate nestin. These factors have been shown to be required for nestin expression in the central nervous system but might be of minor relevance for nestin expression in podocytes because they were not reported to be expressed in the developing kidney.
Nestin is downregulated in an inducible mouse model of glomerular disease.
To gain further insights into the relevance of nestin expression in the kidney, we made use of our recently established inducible mouse model of glomerular disease (33). The animals express the Pax2 transcription factor ectopically in podocytes, which results in podocyte dedifferentiation, glomerular disease with severe proteinuria and high lethality, and, on the molecular level, in reduced expression of Wt1 and its downstream target genes. Immunohistochemical analysis of kidney sections from Pax2 overexpressing mice revealed a dramatic reduction in glomerular nestin expression compared with healthy control animals (Fig. 5A). This reduction in nestin expression was confirmed by quantitative RT-PCR, which showed a decrease in nestin expression to ∼30% of the control levels (Fig. 5B). Whether this reduced nestin expression simply reflects the dedifferentiated phenotype of the podocytes or whether it is a direct effect of the reduced Wt1 expression remains to be clarified, although our in vitro data on Wt1-dependent stimulation of nestin expression support the latter possibility. Our observation of reduced nestin expression in glomerular disease is in apparent contrast to a recent report, which described upregulation of nestin in a rat model of puromycin aminonucleoside-induced nephrosis (42). It is possible that the different models reflect distinct kidney diseases, involving various molecular mechanisms. Unfortunately, the authors did not measure Wt1 expression in their model. However, a definite description of the role of nestin in kidney disease can be expected from nestin studies in patients and animal models with defined inducible upregulation/downregulation of nestin.
Nestin is expressed in the developing epicardium and coronary vessels.
To investigate whether the overlapping expression of nestin and Wt1 is specific for the kidney, we performed double-immunolabeling for both proteins in the developing heart, another known site of Wt1 expression (17, 25, 32, 37). At embryonic day 12.5, nestin and Wt1 shared an overlapping expression pattern mainly in the epicardium. At E15.5, both proteins were coexpressed in the developing coronary vessels (Fig. 6A). The nestin expression in the developing coronary vessels of mouse embryos is in agreement with a previous study showing nestin expression in a variety of developing vessels (24). Interestingly, these authors reported also nestin immunoreactivity in coronary vessels after myocardial infarction, which we had identified as a site of Wt1 expression earlier (32). To further characterize a regulation of nestin by Wt1, we analyzed nestin expression in Wt1 wild-type and knockout hearts at different time points of embryonic development. E15.5 represented the latest possible time point to obtain viable Wt1 knockout embryos (37). Immunohistochemistry revealed a loss of nestin expression in the hearts of Wt1 knockout mice compared with wild-type littermates, suggesting that nestin might be activated by Wt1 also in vivo (Fig. 6B).
In summary, we detected nestin expression in a developmental specific pattern in the kidney and in adult podocytes. As an additional site of nestin immunoreactivity, we identified the epicardium and developing coronary vessels of the heart. In both organs, nestin shared an overlapping expression with the Wilms' tumor suppressor Wt1. Upregulation of nestin in a cell line with inducible Wt1 expression, stimulation of the activity of a nestin enhancer element by cotransfection with Wt1(-KTS), and the loss of nestin in the hearts of Wt1 knockout mice suggest that nestin is regulated by the Wilms' tumor suppressor Wt1.
K. D. Wagner and N. Wagner are recipients of fellowships from Institut National de la Santé et de la Recherche Médicale (INSERM) and European Molecular Biology Organisation (to K. D. Wagner), from the Deutsche Forschungsgemeinschaft (DFG) and INSERM (to N. Wagner). This study was supported in part by grants from the DFG (Scho 634/5–1), the Association pour le Recherche contre le Cancer (#5198), the European Commission (Grant LSHG-CT-2004–005085), and Agence Nationale de Recherches sur le Sida Grant 05135.
The expert technical assistance of A. Richter and I. Grätsch is gratefully acknowledged. The Wt1 expression constructs and the U2OS osteosarcoma cells with inducible Wt1 expression were gifts of D. Haber and C. Englert, respectively. The mouse podocyte cell line was a gift of K. Endlich. Wt1-deficient (Wt1−/−) mouse embryos on the MF1 genetic background were kindly provided by C. Englert.
↵* These authors contributed equally to this article.
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