Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies.

BACKGROUND: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. METHODS: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines' representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. RESULTS: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. CONCLUSION: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression.

Bmp7 drives proximal tubule expansion and determines nephron number in the developing kidney.

The mammalian kidney is composed of thousands of nephrons that are formed through reiterative induction of a mesenchymal-to-epithelial transformation by a population of nephron progenitor cells. The number of nephrons in human kidneys ranges from several hundred thousand to nearly a million, and low nephron number has been implicated as a risk factor for kidney disease as an adult. Bmp7 is among a small number of growth factors required to support the proliferation and self-renewal of nephron progenitor cells, in a process that will largely determine the final nephron number. Once induced, each nephron begins as a simple tubule that undergoes extensive proliferation and segmental differentiation. Bmp7 is expressed both by nephron progenitor cells and the ureteric bud derivative branches that induce new nephrons. Here, we show that, in mice, Bmp7 expressed by progenitor cells has a major role in determining nephron number; nephron number is reduced to one tenth its normal value in its absence. Postnatally, Bmp7 also drives proliferation of the proximal tubule cells, and these ultimately constitute the largest segment of the nephron. Bmp7 appears to act through Smad 1,5,9(8), p38 and JNK MAP kinase. In the absence of Bmp7, nephrons undergo a hypertrophic process that involves p38. Following a global inactivation of Bmp7, we also see evidence for Bmp7-driven growth of the nephron postnatally. Thus, we identify a role for Bmp7 in supporting the progenitor population and driving expansion of nephrons to produce a mature kidney.

GSK3ß and the aging kidney.

Kidney function decreases with age and may soon limit millions of lives as the proportion of the population over 70 years of age increases. Glycogen synthase kinase 3ß (GSK3ß) is involved with metabolism and may have a role in kidney senescence, positioning it as a target for complications from chronic kidney disease. However, different studies suggest GSK3 has contrasting effects. In this issue of the JCI, Fang et al. explored the function of GSK3ß and the interplay with lithium using human tissue and mouse models. Notably, GSK3ß was overexpressed and activated in aging mice, and depleting GSK3ß reduced senescence and glomerular aging. In this Commentary, we explore the similarities and differences between Fang et al. and previous findings by Hurcombe et al. These findings should prompt further study of lithium and other GSK3ß inhibitors as a means of extending glomerular function in individuals with chronic kidney disease.

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro.

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

Epigenetic transcriptional reprogramming by WT1 mediates a repair response during podocyte injury.

In the context of human disease, the mechanisms whereby transcription factors reprogram gene expression in reparative responses to injury are not well understood. We have studied the mechanisms of transcriptional reprogramming in disease using murine kidney podocytes as a model for tissue injury. Podocytes are a crucial component of glomeruli, the filtration units of each nephron. Podocyte injury is the initial event in many processes that lead to end-stage kidney disease. Wilms tumor-1 (WT1) is a master regulator of gene expression in podocytes, binding nearly all genes known to be crucial for maintenance of the glomerular filtration barrier. Using murine models and human kidney organoids, we investigated WT1-mediated transcriptional reprogramming during the course of podocyte injury. Reprogramming the transcriptome involved highly dynamic changes in the binding of WT1 to target genes during a reparative injury response, affecting chromatin state and expression levels of target genes.

The bromodomain inhibitor JQ1 triggers growth arrest and apoptosis in testicular germ cell tumours in vitro and in vivo.

Type II testicular germ cell cancers (TGCT) are the most frequently diagnosed tumours in young men (20-40 years) and are classified as seminoma or non-seminoma. TGCTs are commonly treated by orchiectomy and chemo- or radiotherapy. However, a subset of metastatic non-seminomas (embryonal carcinomas) displays only incomplete remission or relapse and requires novel treatment options. Recent studies have shown effective application of the small-molecule inhibitor JQ1 in tumour therapy, which interferes with the function of 'bromodomain and extraterminal (BET)' proteins. JQ1-treated TGCT cell lines display up-regulation of genes indicative for DNA damage and cellular stress response and induce cell cycle arrest. Embryonal carcinoma (EC) cell lines, which presented as JQ1 sensitive, display down-regulation of pluripotency factors and induction of mesodermal differentiation. In contrast, seminoma-like TCam-2 cells tolerated higher JQ1 concentrations and were resistant to differentiation. ECs xenografted in vivo showed a reduction in tumour size, proliferation rate and angiogenesis in response to JQ1. Finally, the combination of JQ1 and the histone deacetylase inhibitor romidepsin allowed for lower doses and less frequent application, compared with monotherapy. Thus, we propose that JQ1 in combination with romidepsin may serve as a novel therapeutic option for (mixed) TGCTs.

A signaling cascade including ARID1A, GADD45B and DUSP1 induces apoptosis and affects the cell cycle of germ cell cancers after romidepsin treatment.

In Western countries, the incidence of testicular germ cell cancers (GCC) is steadily rising over the last decades. Mostly, men between 20 and 40 years of age are affected. In general, patients suffering from GCCs are treated by orchiectomy and radio- or chemotherapy. Due to resistance mechanisms, intolerance to the therapy or denial of chemo- / radiotherapy by the patients, GCCs are still a lethal threat, highlighting the need for alternative treatment strategies.In this study, we revealed that germ cell cancer cell lines are highly sensitive to the histone deacetylase inhibitor romidepsin in vitro and in vivo, highlighting romidepsin as a potential therapeutic option for GCC patients.Romidepsin-mediated inhibition of histone deacetylases led to disturbances of the chromatin landscape. This resulted in locus-specific histone-hyper- or hypoacetylation. We found that hypoacetylation at the ARID1A promotor caused repression of the SWI/SNF-complex member ARID1A. In consequence, this resulted in upregulation of the stress-sensors and apoptosis-regulators GADD45B, DUSP1 and CDKN1A. RNAi-driven knock down of ARID1A mimicked in parts the effects of romidepsin, while CRISPR/Cas9-mediated deletion of GADD45B attenuated the romidepsin-provoked induction of apoptosis and cell cycle alterations.We propose a signaling cascade involving ARID1A, GADD45B and DUSP1 as mediators of the romidepsin effects in GCC cells.

The Role of Proprotein Convertase Subtilisin/Kexin Type 9 in Nephrotic Syndrome-Associated Hypercholesterolemia.

BACKGROUND: In nephrotic syndrome, damage to the podocytes of the kidney produces severe hypercholesterolemia for which novel treatments are urgently needed. PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as an important regulator of plasma cholesterol levels and therapeutic target. Here, we tested the role of PCSK9 in mediating the hypercholesterolemia of nephrotic syndrome. METHODS: PCSK9 and plasma lipids were studied in nephrotic syndrome patients before and after remission of disease, mice with genetic ablation of the podocyte (Podocyte Apoptosis Through Targeted Activation of Caspase-8, Pod-ATTAC mice) and mice treated with nephrotoxic serum (NTS), which triggers immune-mediated podocyte damage. In addition, mice with hepatic deletion of Pcsk9 were treated with NTS to determine the contribution of PCSK9 to the dyslipidemia of nephrotic syndrome. RESULTS: Patients with nephrotic syndrome showed a decrease in plasma cholesterol and plasma PCSK9 on remission of their disease (P<0.05, n=47-50). Conversely, Pod-ATTAC mice and NTS-treated mice showed hypercholesterolemia and a 7- to 24-fold induction in plasma PCSK9. The induction of plasma PCSK9 appeared to be attributable to increased secretion of PCSK9 from the hepatocyte coupled with decreased clearance. Interestingly, knockout of Pcsk9ameliorated the effects of NTS on plasma lipids. Thus, in the presence of NTS, mice lacking hepatic Pcsk9 showed a 40% to 50% decrease in plasma cholesterol and triglycerides. Moreover, the ability of NTS treatment to increase the percentage of low-density lipoprotein-associated cholesterol (from 9% in vehicle-treated Flox mice to 47% after NTS treatment), was lost in mice with hepatic deletion of Pcsk9 (5% in both the presence and absence of NTS). CONCLUSIONS: Podocyte damage triggers marked inductions in plasma PCSK9, and knockout of Pcsk9 ameliorates dyslipidemia in a mouse model of nephrotic syndrome. These data suggest that PCSK9 inhibitors may be beneficial in patients with nephrotic syndrome-associated hypercholesterolemia.

Reference gene validation for RT-qPCR, a note on different available software packages.

BACKGROUND: An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms. RESULTS: 14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account. CONCLUSIONS: This report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use.

WT1 targets Gas1 to maintain nephron progenitor cells by modulating FGF signals.

Development of the metanephric kidney depends on tightly regulated interplay between self-renewal and differentiation of a nephron progenitor cell (NPC) pool. Several key factors required for the survival of NPCs have been identified, including fibroblast growth factor (FGF) signaling and the transcription factor Wilms' tumor suppressor 1 (WT1). Here, we present evidence that WT1 modulates FGF signaling by activating the expression of growth arrest-specific 1 (Gas1), a novel WT1 target gene and novel modulator of FGF signaling. We show that WT1 directly binds to a conserved DNA binding motif within the Gas1 promoter and activates Gas1 mRNA transcription in NPCs. We confirm that WT1 is required for Gas1 expression in kidneys in vivo. Loss of function of GAS1 in vivo results in hypoplastic kidneys with reduced nephron mass due to premature depletion of NPCs. Although kidney development in Gas1 knockout mice progresses normally until E15.5, NPCs show decreased rates of proliferation at this stage and are depleted as of E17.5. Lastly, we show that Gas1 is selectively required for FGF-stimulated AKT signaling in vitro. In summary, our data suggest a model in which WT1 modulates receptor tyrosine kinase signaling in NPCs by directing the expression of Gas1.

Role of podocyte B7-1 in diabetic nephropathy.

Podocyte injury and resulting albuminuria are hallmarks of diabetic nephropathy, but targeted therapies to halt or prevent these complications are currently not available. Here, we show that the immune-related molecule B7-1/CD80 is a critical mediator of podocyte injury in type 2 diabetic nephropathy. We report the induction of podocyte B7-1 in kidney biopsy specimens from patients with type 2 diabetes. Genetic and epidemiologic studies revealed the association of two single nucleotide polymorphisms at the B7-1 gene with diabetic nephropathy. Furthermore, increased levels of the soluble isoform of the B7-1 ligand CD28 correlated with the progression to ESRD in individuals with type 2 diabetes. In vitro, high glucose conditions prompted the phosphatidylinositol 3 kinase-dependent upregulation of B7-1 in podocytes, and the ectopic expression of B7-1 in podocytes increased apoptosis and induced disruption of the cytoskeleton that were reversed by the B7-1 inhibitor CTLA4-Ig. Podocyte expression of B7-1 was also induced in vivo in two murine models of diabetic nephropathy, and treatment with CTLA4-Ig prevented increased urinary albumin excretion and improved kidney pathology in these animals. Taken together, these results identify B7-1 inhibition as a potential therapeutic strategy for the prevention or treatment of diabetic nephropathy.

Gain-of-function mutations in transient receptor potential C6 (TRPC6) activate extracellular signal-regulated kinases 1/2 (ERK1/2).

Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) gene are a cause of autosomal dominant focal segmental glomerulosclerosis (FSGS). The mechanisms whereby abnormal TRPC6 activity results in proteinuria remain unknown. The ERK1/2 MAPKs are activated in glomeruli and podocytes in several proteinuric disease models. We therefore examined whether FSGS-associated mutations in TRPC6 result in activation of these kinases. In 293T cells and cultured podocytes, overexpression of gain-of-function TRPC6 mutants resulted in increased ERK1/2 phosphorylation, an effect dependent upon channel function. Pharmacologic inhibitor studies implicated several signaling mediators, including calmodulin and calcineurin, supporting the importance of TRPC6-mediated calcium influx in this process. Through medium transfer experiments, we uncovered two distinct mechanisms for ERK activation by mutant TRPC6, a cell-autonomous, EGF receptor-independent mechanism and a non-cell-autonomous mechanism involving metalloprotease-mediated release of a presumed EGF receptor ligand. The inhibitors KN-92 and H89 were able to block both pathways in mutant TRPC6 expressing cells as well as the prolonged elevation of intracellular calcium levels upon carbachol stimulation seen in these cells. However, these effects appear to be independent of their effects on calcium/calmodulin-dependent protein kinase II and PKA, respectively. Phosphorylation of Thr-70, Ser-282, and Tyr-31/285 were not necessary for ERK activation by mutant TRPC6, although a phosphomimetic TRPC6 S282E mutant was capable of ERK activation. Taken together, these results identify two pathways downstream of mutant TRPC6 leading to ERK activation that may play a role in the development of FSGS.

WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing.

Angiogenesis is regulated by the balance of proangiogenic VEGF(165) and antiangiogenic VEGF(165)b splice isoforms. Mutations in WT1, the Wilms' tumor suppressor gene, suppress VEGF(165)b and cause abnormal gonadogenesis, renal failure, and Wilms' tumors. In WT1 mutant cells, reduced VEGF(165)b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth.

WT1-dependent sulfatase expression maintains the normal glomerular filtration barrier.

Paracrine signaling between podocytes and glomerular endothelial cells through vascular endothelial growth factor A (VEGFA) maintains a functional glomerular filtration barrier. Heparan sulfate proteoglycans (HSPGs), located on the cell surface or in the extracellular matrix, bind signaling molecules such as VEGFA and affect their local concentrations, but whether modulation of these moieties promotes normal crosstalk between podocytes and endothelial cells is unknown. Here, we found that the transcription factor Wilms' Tumor 1 (WT1) modulates VEGFA and FGF2 signaling by increasing the expression of the 6-O-endosulfatases Sulf1 and Sulf2, which remodel the heparan sulfate 6-O-sulfation pattern in the extracellular matrix. Mice deficient in both Sulf1 and Sulf2 developed age-dependent proteinuria as a result of ultrastructural abnormalities in podocytes and endothelial cells, a phenotype similar to that observed in children with WT1 mutations and in Wt1(+/-) mice. These kidney defects associated with a decreased distribution of VEGFA in the glomerular basement membrane and on endothelial cells. Collectively, these data suggest that WT1-dependent sulfatase expression plays a critical role in maintaining the glomerular filtration barrier by modulating the bioavailability of growth factors, thereby promoting normal crosstalk between podocytes and endothelial cells.

Copy number variation in patients with disorders of sex development due to 46,XY gonadal dysgenesis.

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.

Expression regulation and function of heparan sulfate 6-O-endosulfatases in the spermatogonial stem cell niche.

Glial cell line-derived neurotrophic factor (GDNF) is a heparan sulfate (HS)-binding factor. GDNF is produced by somatic Sertoli cells, where it signals to maintain spermatogonial stem cells (SSCs) and reproduction. Here, we investigate the roles of extracellular HS 6-O-endosulfatases (Sulfs), Sulf1 and Sulf2, in the matrix transmission of GDNF from Sertoli cells to SSCs. Although Sulfs are not required for testis formation, Sulf deficiency leads to the accelerated depletion of SSCs, a testis phenotype similar to that of GDNF+/- mice. Mechanistically, we show that Sulfs are expressed in GDNF-producing Sertoli cells. In addition, reduced Sulf activity profoundly worsens haplo-deficient GDNF phenotypes in our genetic studies. These findings establish a critical role of Sulfs in promoting GDNF signaling and support a model in which Sulfs regulate the bioavailability of GDNF by enzymatically remodeling HS 6-O-desulfation to release GDNF from matrix sequestration. Further, Sertoli cell-specific transcriptional factor Wilm's tumor 1 (WT1) directly activates the transcription of both Sulf1 and Sulf2 genes. Together, our studies not only identify Sulfs as essential regulators of GDNF signaling in the SSC niche, but also as direct downstream targets of WT1, thus establishing a physiological role of WT1 in Sertoli cells.

Clinical relevance of mutations in the Wilms tumor suppressor 1 gene WT1 and the cadherin-associated protein beta1 gene CTNNB1 for patients with Wilms tumors: results of long-term surveillance of 71 patients from International Society of Pediatric Oncology Study 9/Society for Pediatric Oncology.

BACKGROUND: Mutations in the Wilms tumor (WT) suppressor 1 gene (WT1) and the cadherin-associated protein beta1 gene (CTNNB1) are found predominantly in stromal type WT, defining a genetic subgroup. The clinical relevance of these mutations remains to be determined. METHODS: A long-term follow-up study was performed for 71 patients (International Society of Pediatric Oncology Study 9/Society for Pediatric Oncology; n = 77 tumors) with known molecular genetic status. Eight patients had bilateral disease, including 2 patients with a WT in both kidneys and 5 patients with a WT in 1 kidney and nephrogenic rests (NRs) in the other kidney. The response to preoperative chemotherapy, relapses, metastases, metachronous tumor development, and deaths were evaluated with a median follow-up of 12 years and 4 months. RESULTS: Nineteen patients (n = 24 tumors) had WT1 mutations, and 16 were constitutional mutations. Three patients with germline mutations had second tumor events: Two patients developed a WT in the kidney with NRs 3 years and 11 years after the first tumor; and 1 patient developed second tumors after 2 years, 1 in the kidney with a previous WT and 1 in the kidney with a previous NR. Eighteen of the WT1 mutant tumors were analyzed for CTNNB1 mutations, and all had mutations. A poor volumetric response (progression and <50% reduction) was observed in all patients who had tumors with a WT1 mutation and in 23 of 52 nonmutant tumors. CONCLUSIONS: Patients with WT1 germline mutations had an increased risk for bilateral disease and second tumor events. Therefore, the authors concluded that tumor surveillance until adulthood should be considered. Although tumors with both WT1 and CTNNB1 mutations had a poor volumetric response, there was no significant difference in overall survival in this cohort of patients with and without WT1 mutations.

Characteristics of testicular dysgenesis syndrome and decreased expression of SRY and SOX9 in Frasier syndrome.

Frasier syndrome (FS) is characterized by chronic renal failure in early adulthood, varying degrees of gonadal dysgenesis, and a high risk for gonadal germ cell malignancies, particularly gonadoblastoma. Although it is known to arise from heterozygous splice mutations in intron 9 of the Wilms' tumor gene 1 (WT1), the mechanisms by which these mutations result in gonadal dysgenesis in humans remain obscure. Here we show that a decrease in WT1 + KTS isoforms due to disruption of alternative splicing of the WT1 gene in a FS patient is associated with diminished expression of the transcription factors SRY and SOX9 in Sertoli cells. These findings provide the first confirmation in humans of the results obtained by others in mice. Consequently, Sertoli cells fail to form the specialized environment within the seminiferous tubules that normally houses developing germ cells. Thus, germ cells are unable to fully mature and are blocked at the spermatogonial-spermatocyte stage. Concomitantly, subpopulations of the malignant counterpart of primordial germ cells/gonocytes, the intratubular germ cell neoplasia unclassified type (ITGCN), are identified. Furthermore, dysregulated Leydig cells produce insufficient levels of testosterone, resulting in hypospadias. Collectively, the impaired spermatogenesis, hypospadias and ITGCN comprise part of the developmental disorder known as 'testicular dysgenesis syndrome' (TDS), which arises during early fetal life. The data presented here show that critical levels of WT1 + KTS, SRY and SOX9 are required for normal Sertoli cell maturation, and subsequent normal spermatogenesis. To further study the function of human Sertoli cells in the future, we have established a human cell line.

Impaired glomerular maturation and lack of VEGF165b in Denys-Drash syndrome.

Individuals with Denys-Drash syndrome (DDS) develop diffuse mesangial sclerosis, ultimately leading to renal failure. The disease is caused by mutations that affect the zinc finger structure of the Wilms' tumor protein (WT1), but the mechanisms whereby these mutations result in glomerulosclerosis remain largely obscure. How WT1 regulates genes is likely to be complex, because it has multiple splice forms, binds both DNA and RNA, and associates with spliceosomes. Herein is described that in DDS podocytes, the ratio of both WT1 +KTS isoforms C to D differs considerably from that of normal child and adult control podocytes and more closely resembles fetal profiles. Aside from the delay in podocyte maturation, DDS glomeruli show swollen endothelial cells, reminiscent of endotheliosis, together with incompletely fused capillary basement membranes; a dramatic decrease in collagen alpha4(IV) and laminin beta2 chains; and the presence of immature or activated mesangial cells that express alpha-smooth muscle actin. Because appropriate vascular endothelial growth factor A (VEGF-A) expression is known to be essential for the development and maintenance of glomerular architecture and function, this article addresses the question of whether VEGF-A expression is deregulated in DDS. The data presented here show that DDS podocytes express high levels of the proangiogenic isoform VEGF165, but completely lack the inhibitory isoform VEGF165b. The VEGF165/VEGF165b ratio in DDS resembles that of fetal S-shaped bodies, rather than that of normal child or adult control subjects. The alteration in VEGF-A expression presented here may provide a mechanistic insight into the pathogenesis of DDS.

Frequency and spectrum of cancers in the Peutz-Jeghers syndrome.

BACKGROUND: Although an increased cancer risk in Peutz-Jeghers syndrome is established, data on the spectrum of tumors associated with the disease and the influence of germ-line STK11/LKB1 (serine/threonine kinase) mutation status are limited. EXPERIMENTAL DESIGN: We analyzed the incidence of cancer in 419 individuals with Peutz-Jeghers syndrome, and 297 had documented STK11/LKB1 mutations. RESULTS: Ninety-six cancers were found among individuals with Peutz-Jeghers syndrome. The risk for developing cancer at ages 20, 30, 40, 50, 60, and 70 years was 2%, 5%, 17%, 31%, 60%, and 85%, respectively. The most common cancers represented in this analysis were gastrointestinal in origin, gastroesophageal, small bowel, colorectal, and pancreatic, and the risk for these cancers at ages 30, 40, 50, and 60 years was 1%, 9%, 15%, and 33%, respectively. In women with Peutz-Jeghers syndrome, the risk of breast cancer was substantially increased, being 8% and 31% at ages 40 and 60 years, respectively. Kaplan-Meier analysis showed that cancer risks were similar in Peutz-Jeghers syndrome patients with identified STK11/LKB1 mutations and those with no detectable mutation (log-rank test of difference chi2 = 0.62; 1 df; P = 0.43). Furthermore, the type or site of STK11/LKB1 mutation did not significantly influence cancer risk. CONCLUSIONS: The results from our study provide quantitative information on the spectrum of cancers and risks of specific cancer types associated with Peutz-Jeghers syndrome.