A WT1 Co-regulator Controls Podocyte Phenotype by Shuttling between Adhesion Structures and Nucleus

Abstract

Glomerular podocyte differentiation state is critical for filtration barrier function and is regulated by WT1, a zinc finger transcription factor. A yeast two-hybrid assay identified a novel, WT1-interacting protein (WTIP) that maps to human chromosome 19q13.1, a region with genes linked to familial focal segmental glomerulosclerosis. The domain structure of WTIP is similar to the zyxin subfamily of cytosolic LIM domain-containing proteins, which contain three carboxyl-terminal LIM protein-protein interaction domains and a proline-rich, pre-LIM region with a nuclear export signal. Other LIM domain-containing proteins (zyxin and mouse muscle LIM protein) did not interact with WT1 in two-hybrid assays, and WTIP did not interact with an unrelated transcription factor, LMX1B. WTIP mRNA was detected in cultured podocytes and was developmentally regulated, with expression peaking in mouse kidney at embryonic day 15-16 (E15-E16) in kidney but persisting into adulthood. In situ hybridization demonstrated WTIP expression in developing E15 glomeruli and in cultured podocytes. The partial WTIP clone, which interacted with WTIP in the two-hybrid assay, co-localized with WT1 in nuclei, co-precipitated with WT1, and inhibited WT1-dependent transcriptional activation of the amphiregulin promoter. In contrast, full-length WTIP was excluded from cell nuclei, but after the addition of leptomycin B, an inhibitor of Crm1-mediated nuclear export, it accumulated in the nucleus and co-precipitated with WT1 in whole cell lysates. Epitope-tagged WTIP co-localized with the adaptor protein CD2AP (CMS) in podocyte actin spots and with Mena at cell-cell junctions. We propose that WTIP monitors slit diaphragm protein assembly as part of a multiple protein complex, linking this specialized adhesion junction to the actin cytoskeleton, and shuttles into the nucleus after podocyte injury, providing a mechanism whereby changes in slit diaphragm structure modulate gene expression. Glomerular podocyte differentiation state is critical for filtration barrier function and is regulated by WT1, a zinc finger transcription factor. A yeast two-hybrid assay identified a novel, WT1-interacting protein (WTIP) that maps to human chromosome 19q13.1, a region with genes linked to familial focal segmental glomerulosclerosis. The domain structure of WTIP is similar to the zyxin subfamily of cytosolic LIM domain-containing proteins, which contain three carboxyl-terminal LIM protein-protein interaction domains and a proline-rich, pre-LIM region with a nuclear export signal. Other LIM domain-containing proteins (zyxin and mouse muscle LIM protein) did not interact with WT1 in two-hybrid assays, and WTIP did not interact with an unrelated transcription factor, LMX1B. WTIP mRNA was detected in cultured podocytes and was developmentally regulated, with expression peaking in mouse kidney at embryonic day 15-16 (E15-E16) in kidney but persisting into adulthood. In situ hybridization demonstrated WTIP expression in developing E15 glomeruli and in cultured podocytes. The partial WTIP clone, which interacted with WTIP in the two-hybrid assay, co-localized with WT1 in nuclei, co-precipitated with WT1, and inhibited WT1-dependent transcriptional activation of the amphiregulin promoter. In contrast, full-length WTIP was excluded from cell nuclei, but after the addition of leptomycin B, an inhibitor of Crm1-mediated nuclear export, it accumulated in the nucleus and co-precipitated with WT1 in whole cell lysates. Epitope-tagged WTIP co-localized with the adaptor protein CD2AP (CMS) in podocyte actin spots and with Mena at cell-cell junctions. We propose that WTIP monitors slit diaphragm protein assembly as part of a multiple protein complex, linking this specialized adhesion junction to the actin cytoskeleton, and shuttles into the nucleus after podocyte injury, providing a mechanism whereby changes in slit diaphragm structure modulate gene expression. Podocytes are highly specialized epithelial cells, which synthesize components of glomerular basement membrane, elaborate interdigitating foot processes from adjacent cells that encircle capillaries and are bridged by extracellular proteins of the slit diaphragm. In proteinuric renal diseases, podocytes undergo stereotypic phenotypic simplification into a cuboidal shape, characterized by foot process fusion and retraction and loss of filtration barrier function. Although persistent podocyte dysregulation is associated with glomerular scarring, this phenotype switch is reversible. Appropriate therapy can restore normal podocyte structure and function, suggesting a dynamic, regulated process. Positional cloning and gene targeting have identified the proteins critical for normal podocyte function. However, in the absence of mutations, molecular mechanisms that regulate podocyte phenotype remain unclear. Given its unique microenvironment with exposure to hemodynamic forces and high flow of ultrafiltrate, we speculated that podocytes express intracellular molecules that relay changes in extracellular physical forces or soluble signals to modify cellular structure and function. A number of podocyte proteins are candidate targets as putative regulatory molecules. Normal podocyte differentiation requires the correct temporal expression of specific transcription factor genes (1Quaggin S.E. Microsc. Res. Tech. 2002; 57: 208-211Google Scholar). Of this gene set, we focused on the zinc finger transcription factor WT1 for several reasons. Although originally discovered as a tumor suppressor gene inactivated in a subset of Wilm's tumors, WT1 is essential for normal nephrogenesis. Wt1 null mice lack kidneys (2Kreidberg J.A. Sariola H. Loring J.M. Maeda M. Pelletier J. Housman D. Jaenisch R. Cell. 1993; 74: 679-691Google Scholar), and, when engineered to express a human WT1 transgene, the animals survive but develop mesangial sclerosis or crescentic nephritis (3Guo J.K. Menke A.L. Gubler M.C. Clarke A.R. Harrison D. Hammes A. Hastie N.D. Schedl A. Hum. Mol. Genet. 2002; 11: 651-659Google Scholar), depending on the WT1 expression level. In normal adult animals, WT1 expression is restricted to podocytes, suggesting a role for WT1 in maintenance of podocyte differentiation. In fact, WT1 zinc finger mutations are associated with the re-expression of an immature podocyte phenotype (4Patek C.E. Fleming S. Miles C.G. Bellamy C.O. Ladomery M. Spraggon L. Mullins J. Hastie N.D. Hooper M.L. Hum. Mol. Genet. 2003; 12: 2379-2394Google Scholar) and Denys-Drash and Frasier syndromes (5Hastie N.D. Cell. 2001; 106: 391-394Google Scholar), rare causes of familial glomerulosclerosis. Mice engineered to express these WT1 mutations develop phenotypes that parallel the human syndromes (5Hastie N.D. Cell. 2001; 106: 391-394Google Scholar, 6Hammes A. Guo J. Lutsch G. Leheste J. Landrock D. Ziegler U. Gubler M. Schedl A. Cell. 2001; 106: 319-329Google Scholar, 7Patek C.E. Little M.H. Fleming S. Miles C. Charlieu J.P. Clarke A.R. Miyagawa K. Christie S. Doig J. Harrison D.J. Porteous D.J. Brookes A.J. Hooper M.L. Hastie N.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2931-2936Google Scholar), further documenting that WT1 function is required for a normal podocyte phenotype. Finally, WT1 expression levels decrease in acquired causes of glomerulosclerosis (8Barisoni L. Kriz W. Mundel P. D'Agati V. J. Am. Soc. Nephrol. 1999; 10: 51-61Google Scholar, 9Ohtaka A. Ootaka T. Sato H. Soma J. Sato T. Saito T. Ito S. Am. J. Kidney Dis. 2002; 39: 475-485Google Scholar). The WT1 gene encodes at least 24 different isoforms, with the four major isoforms represented by two alternative splice sites (5Hastie N.D. Cell. 2001; 106: 391-394Google Scholar, 10Haber D.A. Sohn R.L. Buckler A.J. Pelletier J. Call K.M. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9618-9622Google Scholar, 11Lee S.B. Haber D.A. Exp. Cell Res. 2001; 264: 74-99Google Scholar, 12Wagner K.D. Wagner N. Schedl A. J. Cell Sci. 2003; 116: 1653-1658Google Scholar). The first alternative splice site either includes or excludes 17 amino acids encoded by exon 5 and is only expressed in mammals. The function of this domain is not clear, since mice engineered to express WT1 isoforms without exon 5 develop normally and are fertile (13Natoli T.A. McDonald A. Alberta J.A. Taglienti M.E. Housman D.E. Kreidberg J.A. Mol. Cell. Biol. 2002; 22: 4433-4438Google Scholar). The second alternative splice site occurs in intron 9 and includes or excludes three amino acids, KTS. In vitro, WT1 isoforms without KTS (WT1(-KTS)) bind DNA and regulate transcription, whereas those with the KTS (WT1(+KTS)) insertion primarily regulate RNA processing but not gene activity (5Hastie N.D. Cell. 2001; 106: 391-394Google Scholar). WT1 is both a transcriptional repressor and activator (11Lee S.B. Haber D.A. Exp. Cell Res. 2001; 264: 74-99Google Scholar) and regulates genes important in nephron formation including podocalyxin (14Palmer R.E. Kotsianti A. Cadman B. Boyd T. W. Haber D.A. Biol. 2001; 11: Scholar), amphiregulin S.B. K. R. D. J. C. W. Haber D.A. Cell. 1999; Scholar) and (3Guo J.K. Menke A.L. Gubler M.C. Clarke A.R. Harrison D. Hammes A. Hastie N.D. Schedl A. Hum. Mol. Genet. 2002; 11: 651-659Google Scholar). mechanisms of podocyte differentiation we identified proteins that interacted with WT1, a yeast two-hybrid assay with a full-length WT1 exon 5 and the KTS insertion as We a nuclear that regulated WT1 we a novel, cytosolic WT1-interacting protein with a domain structure similar to the zyxin of LIM domain-containing In demonstrated that when the WTIP WT1 transcriptional activity and co-localized with that WTIP the podocyte slit diaphragm and the nucleus in similar to a of cellular molecules that from cell into gene expression. Cell and cells at in and mouse podocytes in the state at and to as P. J. H. Kriz W. R. Exp. Cell Res. Scholar). of J. was into in with the domain to demonstrated that did not and was with and which the activation domain and a kidney from adult mice into the multiple cloning site on and for by a WT1 and In addition to we yeast two-hybrid to and LIM domain-containing proteins, including zyxin and mouse muscle LIM protein We the interaction of mouse WTIP and the transcription factor or a WT1 a that zinc finger the KTS and zinc finger and is in Denys-Drash was by from an in the and a that a after with and the and expression that on and for in the a RNA or RNA was either from cultured podocytes to the and or kidney WTIP expression kidneys from on embryonic day and mice in from day to 9 as B. M. D. M.E. J. Biol. 2003; Scholar). of or of RNA into and to and hybridization with at DNA WTIP was by which the kidney from the two-hybrid Other with the for mouse WT1, and for mouse and in at either mouse or human and by mouse WTIP by of RNA from mouse podocytes was with was with mouse WTIP and and In in situ hybridization was on from which in in and as N. A. S. R.E. D'Agati J.A. M.E. J. Am. Soc. Nephrol. 11: Scholar). a mouse WTIP was into and RNA RNA and and by In situ of and was detected a and at of and for full-length WTIP with of the pre-LIM domain and by cloning the into with fusion to an or to and was a from of cells, or podocytes with the expression to the cells for with at a of in after which was to either after for or in in for cells cultured on for in in and with in for at with cells with in in for at from and mouse and mouse and was a from B. of and with at from to for at or in with on was a and with a with a and and from cells and by and as we have R. S. T. M. J. Scholar, R. B. A. S. S. M. J. 1999; Scholar). cells on and of was to for with protein and at with of with In of cells with and at with and protein by in a for and three with 5 and proteins by in for 5 proteins by to and by a and protein was detected by of and by and into of of fusion proteins expressed and as R. S. T. M. J. Scholar, R. B. A. S. S. M. J. 1999; Scholar). cells with the expression fusion proteins with and from by with as with and at with of or the fusion to or fusion proteins from the WT1 was detected by as S.B. K. R. D. J. C. W. Haber D.A. Cell. 1999; Scholar) amphiregulin to which a WT1 to and a that a with of or cells with a of and and the as activity was in a a of the and of a is required for podocyte but mechanisms its activity and expression are proteins that interact with WT1, we a yeast two-hybrid assay to a kidney from adult mice with a full-length WT1 exon 5 and the KTS insertion after exon 9 (WT1(-KTS)) as A of yeast encoded expressed and and a partial amino was by the expressed in with the activation demonstrated that WTIP and WT1 partial WTIP did not or interact with a yeast domain fusion protein or transcription factor with WT1 in fusion fusion proteins contain only the LIM domain of WTIP and which to the muscle LIM protein with human proteins contain only the LIM domain of WTIP and which to the proteins contain only the LIM domain of WTIP and which to the muscle LIM protein with human in a mouse and human WTIP in the and The partial WTIP identified in the two-hybrid assay to chromosome and was to a human gene on chromosome which with an identified human and mouse expressed which from the and The with the expressed identified in the yeast two-hybrid assay and of full-length human and mouse for a of the full-length mouse and human WTIP protein is in the proteins are and The mouse and human WTIP proteins is restricted to the amino WTIP two amino and a that are not in mouse suggesting these are not critical for WTIP function. The of WTIP to Other of both mouse and human WTIP on the at identified three carboxyl-terminal LIM protein-protein interaction which demonstrated with the zyxin of LIM domain-containing with the fusion protein expression and fusion proteins activation domain and LIM domain-containing proteins muscle LIM protein as protein) or the LIM domains of did not on or express activity assays, the LIM of WTIP and the Denys-Drash WT1 that the interaction was by the region of WTIP and did not the KTS or the zinc finger of WT1 LIM domain from zyxin including a protein similar to and human and mouse WTIP are in The of the are of the and amino are either or suggesting that the LIM domains of these molecules interact with a similar of The LIM domain region of WTIP is to domains protein and the protein with In WTIP a insertion of amino acids in the region the first and second LIM which in and S.E. T. M.C. 2003; Scholar). of WTIP LIM domains with the LIM domains of and protein that on of the amino either or WTIP domain structure from zyxin In to the LIM domain-containing the pre-LIM of WTIP and zyxin from suggesting that this region in this of proteins unique protein-protein The pre-LIM region of WTIP with of amino are on with the zyxin and of amino acids are and mouse WTIP contain two that to domains and in mouse WTIP and and in human mouse and human WTIP contain a domain at the a that these gene from zyxin and specific for WTIP expression was on an RNA WTIP is expressed in a similar to that for WT1 embryonic (2Kreidberg J.A. Sariola H. Loring J.M. Maeda M. Pelletier J. Housman D. Jaenisch R. Cell. 1993; 74: 679-691Google Scholar, 11Lee S.B. Haber D.A. Exp. Cell Res. 2001; 264: 74-99Google Scholar, A.J. Pelletier J. Haber D.A. T. Housman D.E. Mol. Cell. Biol. 1991; 11: Scholar). WTIP are identified in and with expression WT1 and WT1 is required for normal K.D. Wagner N. G. D. Schedl A. C. H. J. 2002; Scholar) as as (11Lee S.B. Haber D.A. Exp. Cell Res. 2001; 264: 74-99Google Scholar) WT1 mutations in are associated with and mesangial sclerosis S. H. V. Nephrol. 2001; Scholar). In WTIP is expressed in and undergo a to epithelial WTIP is a and in human and mouse and in situ hybridization that and podocytes contain WTIP We WTIP expression kidney WTIP mRNA is expressed mouse in a temporal similar to WT1 WTIP mRNA expression is at and into at with to podocytes. In situ hybridization WTIP to and podocytes in developing in E15 kidneys WTIP and WTIP regulate WT1 transcriptional we the of WTIP by WTIP in acids the identified in the yeast two-hybrid assay that only the LIM domain-containing region of co-localized with WT1 in as for a protein that regulates activity of a transcription factor. In contrast, full-length WTIP in the and at sites of cell-cell a that WTIP was discovered as a for a transcription factor. and including protein and from cytosolic to the nucleus D.A. J. S. B. D. M.C. J. Biol. 2001; Scholar, D.A. M.C. J. Cell Biol. Scholar, J. H. Mol. Biol. Cell. 11: Scholar, J. T.A. B. D. Mol. Biol. Cell. 11: Scholar), a process that requires Crm1-mediated nuclear The pre-LIM region of in a nuclear export with a of required for by the export We WTIP expression in cells by both and after with leptomycin B, an inhibitor of Crm1-mediated nuclear export WTIP was in the of cells at the addition of leptomycin B, WTIP accumulated in of cells as as and co-localized with WTIP nuclear was to that in cells with WTIP normally in the and as a cytosolic and the WTIP with WT1 in and WT1-dependent from the yeast two-hybrid we that WT1 and WTIP in cells and from cells with and with or and by with is co-precipitated by both and normal WTIP not with WT1 with WTIP from the nucleus However, WTIP and WT1 did with WTIP when nuclear export is by leptomycin full-length zyxin a zyxin amino acids that to the nucleus D.A. J. S. B. D. M.C. J. Biol. 2001; Scholar) co-precipitated with WT1 assays, a fusion protein or cytosolic domain fusion protein as a WT1 associated with WTIP We WTIP regulated WT1 transcriptional an amphiregulin is an WT1 transcriptional in and its a specific for WT1-dependent transcriptional activation S.B. K. R. D. J. C. W. Haber D.A. Cell. 1999; Scholar). WTIP when in the WT1-dependent transcription activity was and in and cells and with a WT1 expression and the amphiregulin cells with WT1 and an amphiregulin WT1 is is with WT1 and WT1-dependent activity by with cells with WT1 A and did not transcription from In contrast, full-length zyxin regulated WT1-dependent transcriptional that WTIP and WT1 in and that WTIP WT1-dependent transcription its LIM WTIP with of full-length expressed in cultured podocytes. to in podocytes at at cell and in spots WTIP spots of actin which have characterized as sites of assembly D.A. J.A. J. Cell Biol. Scholar), we WTIP co-localized with actin WTIP was not at the of actin but did with actin spots in cells and podocytes CD2AP was originally identified as an protein that the domain of a cell adhesion with the actin and with spots after T. N. Kriz W. K. Am. J. 2001; Scholar). We that CD2AP WTIP and the actin cytoskeleton, since CD2AP domains that interaction with WTIP and four putative that WTIP and CD2AP to actin spots in podocytes with In co-precipitated with CD2AP in In contrast, WTIP did not with focal adhesion or CD2AP have a role in actin assembly and actin is a critical of the podocyte slit a junction D. J. 2001; Scholar, J. Kriz W. M. Mundel P. J. Am. Soc. Nephrol. 11: Scholar), we WTIP was in to podocyte Mena as a Mena and a protein to a role in and of actin at developing V. C. M. Cell. Scholar, V. Cell Biol. 2001; Scholar). We podocytes to and cell-cell junctions. in WTIP and Mena at which have characterized as developing V. C. M. Cell. Scholar, V. Cell Biol. 2001; Scholar). Mena is to focal but WTIP did not with Mena in these adhesion with lack of of WTIP and and that WTIP to part of a that actin assembly to the slit diaphragm Podocytes from the after a differentiation to a highly specialized phenotype. In vitro, and human that podocyte dysregulation can glomerular W. Microsc. Res. Tech. 2002; 57: Scholar). In proteinuric diseases, of the podocytes undergo changes Microsc. Res. Tech. 2002; 57: Scholar). The actin into a the to the slit diaphragm are and the podocyte a cuboidal Microsc. Res. Tech. 2002; 57: Scholar). a molecular in both human and this is associated with changes in membrane, and nuclear podocyte differentiation expression. Given its unique microenvironment with exposure to hemodynamic forces and high flow of ultrafiltrate, the podocyte to to changes in physical forces or soluble processes that of actin in the and changes in gene expression in the number of molecules are to and nucleus to regulate gene expression. We speculated that specific molecules from the filtration barrier to the nucleus to the podocyte to regulate its differentiation state in to which regulate podocyte cell changes in gene expression. from mice and with syndromes including glomerular have identified several transcription required for podocyte and including WT1 (1Quaggin S.E. Microsc. Res. Tech. 2002; 57: 208-211Google Scholar, N.D. Cell. 2001; 106: 391-394Google Scholar). proteins that regulate podocyte a kidney was in a two-hybrid assay with WT1 as We identified a that three LIM protein-protein interaction domains and is similar to the zyxin of adhesion molecules. The expression of WTIP and WT1 WTIP a nuclear export and export is inhibited by leptomycin B. WTIP is in the it with WT1 and WT1-dependent transcriptional activation of the amphiregulin promoter. WTIP to a of podocyte which WT1-dependent gene that regulate WT1 function remain WT1 with a number of proteins (11Lee S.B. Haber D.A. Exp. Cell Res. 2001; 264: 74-99Google Scholar). WT1 and the transcriptional protein interact the first two zinc of WT1 and the domain of to the interaction of the amphiregulin W. S.B. R. Haber D.A. J. Biol. 2001; Scholar). can or WT1-dependent transcription by with either the exon 5 region or the zinc finger D.J. V. B. 2001; Scholar, J. S. C. Haber D.A. T. Mol. Cell. Biol. Scholar). a domain-containing and of proteins transcription by WT1 J. N. H. T. J. Biol. Scholar, V. P. A.J. J. Biol. Scholar). LIM domain-containing with WT1 to modulate transcription of genes important in differentiation P. T. D. C. R. 2002; Scholar). protein) was identified yeast two-hybrid and in nuclear but its function is Hastie N.D. Hum. Mol. Genet. Scholar). Although persistent expression of WT1 in podocyte that podocyte differentiation requires transcription of WT1-dependent of the WT1 protein WT1 activity is regulated in the WTIP a The domain structure of WTIP it is a in the absence of WTIP co-localized with CD2AP in actin sites of actin assembly in cells, and with Mena at sites of developing cell-cell that WTIP is for normal podocyte differentiation by proteins that cell-cell foot processes and that WTIP is excluded from to signals the two cellular carboxyl-terminal LIM domains and and sites the protein-protein interaction a domain structure to the zyxin of LIM domain-containing both human and WTIP and the S.E. T. M.C. 2003; Scholar) and is characterized by amino and three LIM protein-protein interaction The LIM domain is a zinc finger protein interaction and proteins LIM domains cell and and R. Genet. Scholar, Scholar). and LIM domain-containing proteins from sites of cell to the nucleus and can regulate cell differentiation state J. H. Mol. Biol. Cell. 11: Scholar, J. T.A. B. D. Mol. Biol. Cell. 11: Scholar, 2001; Scholar). In addition to proteins to the nucleus and bind to transcription including and human K. J. Scholar, M. Scholar, A. S. Cell. Mol. Sci. 1999; Scholar). that cell the podocyte slit function as molecules that can to the nucleus and regulate cell to extracellular and have to the molecular of the filtration which requires of podocyte foot processes and assembly of and specialized cell-cell the slit for normal function. The slit diaphragm is a junction that proteins that have discovered by cloning and and by protein an is to a major of the slit diaphragm M. U. M. J. P. H. V. T. M. R. C.E. L. C. A. K. Mol. Cell. Scholar). which is similar to a structure with both the and domains in the N. S. H. A. K. Gubler M.C. P. C. Genet. Scholar). CD2AP was originally identified as an adaptor protein that with the domain of a cell adhesion but mice CD2AP develop glomerulosclerosis J. V. A. M.L. 1999; Scholar). in human CD2AP have in and familial glomerulosclerosis J.M. H. G. 2003; Scholar). three proteins to the slit diaphragm. The that CD2AP with its domain J. R. Mundel P. Am. J. 2001; Scholar). The of with both and CD2AP in K. M. J. C. Kriz W. Mundel P. J. 2001; Scholar, M. B. G. T. J. Biol. 2001; Scholar) and M. B. G. T. J. Biol. 2001; Scholar). In addition to these proteins, the slit diaphragm a which with protein and but not a of J. Kriz W. M. Mundel P. J. Am. Soc. Nephrol. 11: Scholar). Although physical the and podocyte are to a in the assembly of these molecules D. J. 2001; Scholar). a critical role in formation and maintenance of these in is restricted to podocyte foot processes D. Scholar). The human of was discovered as a for the focal adhesion protein to and with actin and interact with V. Cell Biol. 2001; Scholar), suggesting that actin is essential for the podocyte foot process and adhesion process retraction is the podocyte to and is characterized both in and in by actin J. 2002; Scholar), further a role for actin as a of podocyte foot process which encodes the actin protein was identified as a candidate gene on chromosome from three with focal glomerulosclerosis. with the actin and important in the filtration since mutations in focal glomerulosclerosis J.M. H. Genet. Scholar). mutations a phenotype similar to mutations in and podocalyxin that have in or suggesting that actin is a critical to assembly of podocyte slit diaphragm proteins D. J. 2001; Scholar). of the actin with junction assembly V. C. M. Cell. Scholar, V. Cell Biol. 2001; Scholar). that WTIP is expressed in podocytes in and in WTIP with CD2AP and a protein associated with cell and in to podocyte actin WTIP a nuclear export and its nuclear export is inhibited by leptomycin B. WTIP is in the WTIP with WT1 and WT1-dependent transcriptional activation of the amphiregulin promoter. on these we the In normal WTIP is part of a in podocyte foot process and the to proteins by actin injury, WTIP into the it WT1-dependent gene in dysregulation podocyte phenotype. of WTIP from its cytosolic of slit diaphragm proteins and actin of foot process In WTIP to a of podocyte which is part of a of cell adhesion molecules that extracellular into gene expression by and We J. Pelletier for the mouse WT1