Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70 - PubMed (original) (raw)

. 1998 Apr 15;12(8):1108-20.

doi: 10.1101/gad.12.8.1108.

C Englert, G Zheng, S B Lee, J Wong, D P Harkin, J Bean, R Ezzell, A J Garvin, R T McCluskey, J A DeCaprio, D A Haber

Affiliations

Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70

S Maheswaran et al. Genes Dev. 1998.

Abstract

The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Colocalization of WT1 and Hsp70 is evident within podocytes of the developing kidney, and Hsp70 is recruited to the characteristic subnuclear clusters that contain WT1. The amino-terminal transactivation domain of WT1 is required for binding to Hsp70, and expression of that domain itself is sufficient to induce expression of Hsp70 through the heat shock element (HSE). Substitution of a heterologous Hsp70-binding domain derived from human DNAJ is sufficient to restore the functional properties of a WT1 protein with an amino-terminal deletion, an effect that is abrogated by a point mutation in DNAJ that reduces binding to Hsp70. These observations indicate that Hsp70 is an important cofactor for the function of WT1, and suggest a potential role for this chaperone during kidney differentiation.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Coimmunoprecipitation of WT1 and Hsp70. (A) (Left) Immunoprecipitation of metabolically labeled cellular lysates from Cos-7 cells transfected with an HA-epitope tagged WT1 construct or vector, with the 12-CA-5 monoclonal antibody directed against the HA-epitope. (Middle) Immunoprecipitation of labeled lysates from U2OS cells with tetracycline-regulated expression of WT1. WT1 expression was induced by withdrawal of tetracycline and extracts were immunoprecipitated with the polyclonal antibody WTc8, directed against the amino terminus of WT1, or its preimmune serum. (Right) U2OS cells were grown in the presence or absence of tetracycline, followed by metabolic labeling and immunoprecipitation with the monoclonal antibody C19, directed against the carboxyl terminus of WT1. The arrow denotes the migration position of the coprecipitated band of ∼65 kD. (B) Amino acid sequence of two peptides derived from microsequencing of proteolytic products, showing identity with Hsp70.1 and Hsp70.2. Parentheses denote probable, but not definitive residues. (C) hsp70–Western blot of anti-WT1 immunoprecipitates, derived from U2OS cells with inducible WT1 grown in the presence or absence of tetracycline. Cellular lysate (formula image the amount immunoprecipitated) was analyzed directly to demonstrate the migration position of native Hsp70.

Figure 1

Figure 1

Coimmunoprecipitation of WT1 and Hsp70. (A) (Left) Immunoprecipitation of metabolically labeled cellular lysates from Cos-7 cells transfected with an HA-epitope tagged WT1 construct or vector, with the 12-CA-5 monoclonal antibody directed against the HA-epitope. (Middle) Immunoprecipitation of labeled lysates from U2OS cells with tetracycline-regulated expression of WT1. WT1 expression was induced by withdrawal of tetracycline and extracts were immunoprecipitated with the polyclonal antibody WTc8, directed against the amino terminus of WT1, or its preimmune serum. (Right) U2OS cells were grown in the presence or absence of tetracycline, followed by metabolic labeling and immunoprecipitation with the monoclonal antibody C19, directed against the carboxyl terminus of WT1. The arrow denotes the migration position of the coprecipitated band of ∼65 kD. (B) Amino acid sequence of two peptides derived from microsequencing of proteolytic products, showing identity with Hsp70.1 and Hsp70.2. Parentheses denote probable, but not definitive residues. (C) hsp70–Western blot of anti-WT1 immunoprecipitates, derived from U2OS cells with inducible WT1 grown in the presence or absence of tetracycline. Cellular lysate (formula image the amount immunoprecipitated) was analyzed directly to demonstrate the migration position of native Hsp70.

Figure 1

Figure 1

Coimmunoprecipitation of WT1 and Hsp70. (A) (Left) Immunoprecipitation of metabolically labeled cellular lysates from Cos-7 cells transfected with an HA-epitope tagged WT1 construct or vector, with the 12-CA-5 monoclonal antibody directed against the HA-epitope. (Middle) Immunoprecipitation of labeled lysates from U2OS cells with tetracycline-regulated expression of WT1. WT1 expression was induced by withdrawal of tetracycline and extracts were immunoprecipitated with the polyclonal antibody WTc8, directed against the amino terminus of WT1, or its preimmune serum. (Right) U2OS cells were grown in the presence or absence of tetracycline, followed by metabolic labeling and immunoprecipitation with the monoclonal antibody C19, directed against the carboxyl terminus of WT1. The arrow denotes the migration position of the coprecipitated band of ∼65 kD. (B) Amino acid sequence of two peptides derived from microsequencing of proteolytic products, showing identity with Hsp70.1 and Hsp70.2. Parentheses denote probable, but not definitive residues. (C) hsp70–Western blot of anti-WT1 immunoprecipitates, derived from U2OS cells with inducible WT1 grown in the presence or absence of tetracycline. Cellular lysate (formula image the amount immunoprecipitated) was analyzed directly to demonstrate the migration position of native Hsp70.

Figure 2

Figure 2

Physical association of endogenous WT1 and Hsp70. (A) Immunoprecipitation-Western analysis of extracts from embryonic rat kidney cells that express endogenous WT1. Equal amounts of cellular lysates were immunoprecipitated with either anti-WT1 antibody C19 or a nonspecific control antibody (against c-rel), followed by immunoblotting analysis with anti-Hsp70 antibody. Cellular lysate (1/20 the amount immunoprecipitated) was analyzed directly to show the migration position of native Hsp70. (B) Immunoprecipitation–Western analysis of lysates from sporadic Wilms tumor specimens. The tumors, denoted by initials, are known to express wild-type WT1. Equal amounts of cellular lysates were immunoprecipitated with anti-WT1 antibody WTc8, followed by immunoblotting with antibody against Hsp70. Cellular lysate from tumor GS (1/20 the amount immunoprecipitated) was analyzed directly by immunoblotting. (C) Immunoprecipitation of radiolabeled lysates from embryonic rat kidney cells to demonstrate the relative proportion of WT1 and Hsp70 that are coimmunoprecipitated with each other. Equal amounts of cellular lysates were immunoprecipitated with either anti-WT1 antibody C19 or anti-Hsp70 antibody. The amount of total cellular WT1 directly immunoprecipitated with C19 was compared with the amount coimmunoprecipitated with Hsp70 antibody; the amount of total cellular Hsp70 immunoprecipitated directly was compared with the amount coimmunoprecipitated by use of anti-WT1 antibody C19.

Figure 2

Figure 2

Physical association of endogenous WT1 and Hsp70. (A) Immunoprecipitation-Western analysis of extracts from embryonic rat kidney cells that express endogenous WT1. Equal amounts of cellular lysates were immunoprecipitated with either anti-WT1 antibody C19 or a nonspecific control antibody (against c-rel), followed by immunoblotting analysis with anti-Hsp70 antibody. Cellular lysate (1/20 the amount immunoprecipitated) was analyzed directly to show the migration position of native Hsp70. (B) Immunoprecipitation–Western analysis of lysates from sporadic Wilms tumor specimens. The tumors, denoted by initials, are known to express wild-type WT1. Equal amounts of cellular lysates were immunoprecipitated with anti-WT1 antibody WTc8, followed by immunoblotting with antibody against Hsp70. Cellular lysate from tumor GS (1/20 the amount immunoprecipitated) was analyzed directly by immunoblotting. (C) Immunoprecipitation of radiolabeled lysates from embryonic rat kidney cells to demonstrate the relative proportion of WT1 and Hsp70 that are coimmunoprecipitated with each other. Equal amounts of cellular lysates were immunoprecipitated with either anti-WT1 antibody C19 or anti-Hsp70 antibody. The amount of total cellular WT1 directly immunoprecipitated with C19 was compared with the amount coimmunoprecipitated with Hsp70 antibody; the amount of total cellular Hsp70 immunoprecipitated directly was compared with the amount coimmunoprecipitated by use of anti-WT1 antibody C19.

Figure 3

Figure 3

Colocalization of WT1 and Hsp70 in cultured cells. (A) Immunofluorescence analysis of U2OS cells with tetracycline-regulated expression of the wild-type isoform WT1(−KTS), a truncated mutant lacking the carboxy-terminal zinc finger domain (WT1–del Z), and the characteristic chromosomal translocation product identified in Desmoplastic Small Round Cell Tumor (the EWS–WT1(−KTS) chimera, comprised of the amino-terminal domain of the EWS fused to the carboxy-terminal zinc finger domain of WT1). Cells were grown in the presence or absence of tetracycline and stained by use of antibodies against the amino terminus of WT1 (WT1 (+ and − KTS), the HA epitope tag (EWS–WT1), or against Hsp70. (B) Confocal imaging of U2OS cells with inducible expression of WT1–delZ, following staining with rhodamine-conjugated anti-WT1 and fluorescein-conjugated anti-Hsp70 antibodies. The yellow signal in the merged image identifies precise overlap between red and green signals. Bar, 10 μm.

Figure 3

Figure 3

Colocalization of WT1 and Hsp70 in cultured cells. (A) Immunofluorescence analysis of U2OS cells with tetracycline-regulated expression of the wild-type isoform WT1(−KTS), a truncated mutant lacking the carboxy-terminal zinc finger domain (WT1–del Z), and the characteristic chromosomal translocation product identified in Desmoplastic Small Round Cell Tumor (the EWS–WT1(−KTS) chimera, comprised of the amino-terminal domain of the EWS fused to the carboxy-terminal zinc finger domain of WT1). Cells were grown in the presence or absence of tetracycline and stained by use of antibodies against the amino terminus of WT1 (WT1 (+ and − KTS), the HA epitope tag (EWS–WT1), or against Hsp70. (B) Confocal imaging of U2OS cells with inducible expression of WT1–delZ, following staining with rhodamine-conjugated anti-WT1 and fluorescein-conjugated anti-Hsp70 antibodies. The yellow signal in the merged image identifies precise overlap between red and green signals. Bar, 10 μm.

Figure 4

Figure 4

Colocalization of WT1 and Hsp70 in developing glomerular podocytes. Immunohistochemical analysis of sections from a 13-week human kidney, by use of antibodies against WT1 and Hsp70. Low power (200×) reveals developing glomeruli, in which podocytes are seen as a ring of peripheral cells that stain intensely for both WT1 and Hsp70. The mesangial cells, renal tubular cells, and stroma are negative. At higher power (630X), staining for both WT1 and Hsp70 is seen to be restricted to the nuclei of podocytes, and exhibit a speckled pattern.

Figure 5

Figure 5

Transcriptional activation of hsp70 by WT1. (A) Induction of Hsp70 protein following expression of WT1. U2OS cells with inducible expression of WT1 were grown in the presence or absence (24 hr) of tetracycline, and equal amounts of cellular lysates were analyzed by immunoblotting with antibody against Hsp70. Induction of WT1 is shown at bottom. (B) Induction of hsp70.1 mRNA by WT1. Northern blot analysis of U2OS cells with inducible WT1, EWS–WT1, or empty vector, following growth in the presence or absence (24 hr) of tetracycline. A gene-specific probe was derived from the 3′ untranslated region of human hsp70.1. (Middle) Reprobing of the blot with a WT1 cDNA to confirm inducible expression of WT1 and the EWS–WT1 chimera; (bottom) a GAPDH loading control. (C) Transcriptional activation of the hsp70 promoter by WT1. U2OS cells were transfected with CMV-driven WT1(−KTS) or empty vector, along with reporter constructs, followed by determination of CAT activity. The respective fragments of the hsp70 promoter reporter are shown (bottom), including the primary HSE and CCAAT regulatory elements. The HSE–CAT reporter contains multimerized HSE sites. The fold induction of CAT activity was determined by scintillation counting.

Figure 6

Figure 6

Identification of amino-terminal WT1 domain required for association with Hsp70. (A) Schematic representation of WT1 deletion constructs. The chimeric constructs DNAJ–WT1 encodes the 78 amino acid J domain of human DNAJ (HSJ1). H42Q–DNAJ–WT1 contains a substitution of glutamine for histidine within the critical HPD residues required for association with Hsp70 (Wall et al. 1994; Tsai and Douglas 1996). (B) Coimmunoprecipitation of Hsp70 with truncated WT1 proteins. Cos-7 cells were transfected with CMV-driven constructs, followed by immunoprecipitation of radiolabeled lysates with antibody 12-CA-5 against the HA epitope. Comparable expression of the WT1 constructs is demonstrated, along with coimmunoprecipitation of Hsp70 with all WT1 deletion constructs except Δ6-180. (C) Coimmunoprecipitation of Hsp70 with DNAJ–WT1, but not H42Q–DNAJ–WT1. Cos-7 cells were transfected with CMV driven constructs encoding wild-type WT1 or the WT1–DNAJ chimerae, followed by coimmunoprecipitation with antibody 12-CA-5.

Figure 6

Figure 6

Identification of amino-terminal WT1 domain required for association with Hsp70. (A) Schematic representation of WT1 deletion constructs. The chimeric constructs DNAJ–WT1 encodes the 78 amino acid J domain of human DNAJ (HSJ1). H42Q–DNAJ–WT1 contains a substitution of glutamine for histidine within the critical HPD residues required for association with Hsp70 (Wall et al. 1994; Tsai and Douglas 1996). (B) Coimmunoprecipitation of Hsp70 with truncated WT1 proteins. Cos-7 cells were transfected with CMV-driven constructs, followed by immunoprecipitation of radiolabeled lysates with antibody 12-CA-5 against the HA epitope. Comparable expression of the WT1 constructs is demonstrated, along with coimmunoprecipitation of Hsp70 with all WT1 deletion constructs except Δ6-180. (C) Coimmunoprecipitation of Hsp70 with DNAJ–WT1, but not H42Q–DNAJ–WT1. Cos-7 cells were transfected with CMV driven constructs encoding wild-type WT1 or the WT1–DNAJ chimerae, followed by coimmunoprecipitation with antibody 12-CA-5.

Figure 7

Figure 7

Inhibition of cellular proliferation by WT1 requires association with Hsp70. (A) Inhibition of colony formation in U2OS and Saos-2 cells following transfection of wild-type WT1, the amino-terminal truncation WT1–Δ6–180, DNAJ–WT1, H42Q–DNAJ–WT1 (encoding a point mutation within the DNAJ domain), and DNAJ–WT1–delZ (encoding a truncation of the WT1 DNA binding domain). All WT1 constructs (except DNAJ–WT1–delZ) encoded the (−KTS) splicing variant, with an uninterrupted DNA binding domain, which has been linked to inhibition of cellular proliferation in these osteosarcoma cells. Cells were cotransfected with a construct encoding puromycin resistance and drug resistant colonies were counted after 14 days (U2OS) or 21 days (Saos-2) in culture. Standard deviations are given. (B) Absence of correlation between transcriptional repression or promoter-reporter constructs and inhibition of colony formation by WT1 variants. U2OS cells were transfected with WT1 and DNAJ–WT1 constructs, along with either the GC-rich EGR1–CAT or the TC-rich EGFR–CAT promoter reporters. Representative experiments are shown, with transcriptional repression quantitated by scintillation counting. (C) G1 phase cell cycle arrest induced by wild-type WT1 and DNAJ–WT1. Saos-2 cells were transiently transfected with WT1 and DNAJ–WT1 constructs, along with a construct encoding the cell surface marker CD20. CD20-expressing transfectants were identified by FACS analysis, and their cell cycle distribution was determined by staining with propidium iodide. A representative experiment is shown. (D) Induction of p21 by wild-type WT1 and DNAJ–WT1. Saos-2 cells were transiently transfected with constructs encoding WT1 and DNAJ–WT1 variants. Cellular extracts were isolated 24 hr after transfection, and equal amounts of lysates were analyzed by immunoblotting by use of antibody against p21. Saos-2 cells have a deletion of endogenous p53 and express low levels of endogenous p21; their high transfection efficiency makes it possible to analyze the induction of the native p21 gene following transient transfection of expression constructs. As a control, induction of p21 is shown following transient transfection of CMV-driven p53 (1 μg).

Similar articles

Cited by

References

    1. Abravaya K, Myers M, Murphy S, Morimoto R. The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression. Genes & Dev. 1992;6:1153–1164. - PubMed
    1. Agoff SN, Hou J, Linzer DI, Wu B. Regulation of the human hsp70 promoter by p53. Science. 1993;259:84–87. - PubMed
    1. Bhan AK. Immunoperoxidase. In: Colvin RB, Bhan AK, McCluskey RT, editors. Diagnostic immunopathology. 2nd edition. NY: Raven Press; 1995. pp. 711–723.
    1. Bonnycastle LL, Yu CE, Hunt CR, Trask BJ, Clancy KP, Weber JL, Patterson D, Schellenberg GD. Cloning, sequencing, and mapping of the human chromosome 14 heat shock protein gene (HSPA2) Genomics. 1994;23:85–93. - PubMed
    1. Campbell K, Mullane K, Aksoy I, Stubdal H, Zalvide J, Pipas J, Silver P, Roberts T, Schaffhausen B, DeCaprio J. DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication. Genes & Dev. 1997;11:1098–1110. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources