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Biomedical subjects

B Knebelmann

Publications and source records attributed to B Knebelmann.

At least 19 recordsLinked to original sources
PubMed2001

VHL tumor suppressor regulates Cl-/HCO3- exchange and Na+/H+ exchange activities in renal carcinoma cells.

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are thought to play a critical role in the pathogenesis of both sporadic and VHL disease-associated clear-cell renal carcinomas (RCC). Differential display-PCR identified the AE2 anion exchanger as a candidate VHL target gene. AE2 mRNA and polypeptide levels were approximately threefold higher in 786-O VHL cells than in 786-O Neo cells. In contrast, Cl(-)/HCO(3)(-) exchange activity in 786-O VHL cells was 50% lower than in 786-O Neo cells. Since resting intracellular pH (pH(i)) values were indistinguishable, we postulated that Na(+)/H(+) exchange activity (NHE) might be similarly reduced in 786-O VHL cells. NHE-mediated pH(i) recovery from acid load was less than 50% that in 786-O Neo cells, whereas hypertonicity-stimulated, amiloride-sensitive NHE was indistinguishable in the two cell lines. The NHE3 mRNA level was higher in 786-O VHL than 786-O Neo cells, but NHE1 mRNA levels did not differ. AE2 and NHE3 are the first transcripts reported to be upregulated by pVHL. Elucidation of mechanisms responsible for downregulation of both ion exchange activities will require further investigation.

Ammonium Chloride↗
PubMed2001

Granulomatous renal disease in a patient with common variable immunodeficiency.

Common variable immunodeficiency (CVID), the most common cause of primary hypogammaglobulinemia, is characterized by a decreased serum immunoglobulin level, recurrent infections, and the occurrence of various autoimmune diseases. Granulomatous disease has been reported previously in several patients with CVID, with granuloma occurring in the lymph nodes, spleen, liver, central nervous system, and bone marrow. We report the first published case of renal granulomatous disease in a CVID patient presenting with subacute renal failure. Renal function partially recovered after corticosteroid treatment and intravenous immunoglobulin infusions. The pathogenesis of granulomatous disease in CVID is unclear but may involve monocyte and T-cell abnormalities.

Adult↗
PubMed1999

An important von Hippel-Lindau tumor suppressor domain mediates Sp1-binding and self-association.

VHL is the causative gene for both von Hippel-Lindau (VHL) disease and sporadic clear-cell renal cancer. We showed earlier that VHL downregulates vascular endothelial growth factor transcription by directly binding and inhibiting the transcriptional activator Sp1. We have now mapped the VHL Sp1-binding domain to amino acids 96-122. The 96-122 domain is disproportionately affected by substitution mutations, which interfere with the VHL-Sp1 interaction. Deletion of the 96-122 domain prevents VHL effects on Sp1 DNA binding and on VHL target gene expression, indicating the domain contributes importantly to VHL tumor suppressor activity. Nevertheless, prevention of the VHL-Sp1 interaction only partially abrogates VHL's transcriptional repressor activity, supporting the existence of VHL transcriptional effectors in addition to Sp1. VHL also directly interacts with the Sp1 zinc fingers and self-associates via the 96-122 domain, which furthermore suggest the domain may bind other metalloproteins and contribute to VHL dominant-negative effects.

Amino Acid Substitution↗
PubMed1999

Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells.

Angiostatin which contains the first four kringle domains of plasminogen has been documented to be a potent inhibitor of angiogenesis. More recently, another kringle structure within plasminogen but outside angiostatin, known as kringle 5 (K5), was found to inhibit endothelial cell proliferation and migration. Here, we report the cloning and expression of mouse kringle 5 (rK5) in a bacterial expression system. The protein was purified to homogeneity using a Ni-NTA column. rK5 inhibited both proliferation and migration of endothelial cells with ED50's of 10 nM and < 500 nM, respectively. In addition, we show for the first time that rK5 causes cell cycle arrest and apoptosis, shedding further insight into rK5's mechanism of action. Finally, we show that these actions are endothelial cell specific.

Amino Acid Sequence↗
PubMed1999

Transforming growth factor beta1 is a target for the von Hippel-Lindau tumor suppressor and a critical growth factor for clear cell renal carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in patients with VHL disease and in the majority of patients with sporadic clear cell renal carcinoma (RCC). Overexpression of transforming growth factor (TGF) beta1 has been observed in patients with several cancers, including RCCs, with serum and urine levels correlating inversely with prognosis. We have demonstrated that the VHL tumor suppressor gene product represses TGF-beta1 mRNA and protein levels (approximately 3-4-fold) in 786-O RCC cells by decreasing the TGF-beta1 mRNA half-life. Exogenously added TGF-beta1 did not suppress the growth of 786-O cells in vitro, nor did the addition of neutralizing antibody (Ab) against TGF-beta have any effect. Indeed, 786-O cells were found to express no TGF-beta type II receptor protein, thus allowing them to escape from the negative growth control of TGF-beta1. In contrast to the in vitro data, neutralizing Ab to TGF-beta inhibited tumorigenesis and, in some cases, regressed established 786-O tumors in athymic mice. Immunohistochemistry for von Willebrand's factor revealed a 3-4-fold lower tumor microvessel count in the mice treated with TGF-beta Ab compared to controls, suggesting that the Ab was inhibiting angiogenesis. Our findings indicate that TGF-beta1 is a novel target for the VHL tumor suppressor and that antagonizing its paracrine action may provide novel avenues for treatment of RCCs as well as other tumors that secrete TGF-beta1.

Adenocarcinoma, Clear Cell↗
PubMed1999

Endostatin induces endothelial cell apoptosis.

Endostatin, a carboxyl-terminal fragment of collagen XVIII, has been shown to regress tumors in mice. In this study, we have analyzed the mechanism of endostatin action on endothelial cells and nonendothelial cells. Endostatin treatment of cow pulmonary artery endothelial cells caused apoptosis, as demonstrated by three methods, annexin V-fluorescein isothiocyanate staining, caspase 3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling assay. Moreover, addition of endostatin led to a marked reduction of the Bcl-2 and Bcl-XL anti-apoptotic protein, whereas Bax protein levels were unaffected. These effects were not seen in several nonendothelial cells. Collectively, these findings provide important mechanistic insight into endostatin action.

Animals↗
PubMed1999

Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin.

Based on a homology search with endostatin, the C-terminus 185 aa of collagen XVIII, we report the cloning, expression, and antiangiogenic activity of a 22 kDa human collagen XV fragment, that we have named restin. Restin was expressed in the prokaryotic pET expression system. We have shown that restin inhibits the migration of endothelial cells in vitro but has no effect on the proliferation of these cells. A polyclonal antibody raised against endostatin cross-reacted with restin. Systemic administration of restin suppressed the growth of tumors in a xenograft renal carcinoma model.

Amino Acid Sequence↗
PubMed1998

Transforming growth factor alpha is a target for the von Hippel-Lindau tumor suppressor.

The von Hippel-Lindau (VHL) tumor suppressor gene has a critical role in the pathogenesis of clear cell renal cell carcinoma (RCC), because VHL mutations have been found in both VHL disease-associated and sporadic RCC. Overexpression of transforming growth factor (TGF)-alpha has been observed in numerous RCC tumors and cell lines, and TGF-alpha has been demonstrated to support RCC cell growth through an autocrine loop. We demonstrate here that VHL substantially decreases TGF-alpha message and protein by shortening TGF-alpha mRNA half-life. By Northern analysis TGF-alpha mRNA steady-state levels were suppressed 5-fold in permanent 786-0 RCC cell lines expressing wild-type VHL compared with 786-0 cells expressing an empty vector or a mutant VHL protein lacking COOH-terminal residues 116-213 (deltaVHL). By Western analysis, VHL also substantially down-regulated the unprocessed, cell-associated Mr 20,000 TGF-alpha protein. Moreover, secreted TGF-alpha was undetectable in VHL-expressing cells. In contrast, VHL did not down-regulate the TGF-alpha receptor, epidermal growth factor receptor, either at the mRNA or protein level. Nuclear run-on in vitro transcription experiments in 786-0 cells showed that VHL did not affect transcriptional control of the endogenous TGF-alpha gene. However, actinomycin D experiments revealed a long TGF-alpha mRNA half-life in 786-0 cells that was significantly decreased by wild-type VHL but not by deltaVHL. We have, therefore, identified TGF-alpha, an important growth factor for RCC, as a new target gene for VHL and demonstrated that VHL acts by decreasing TGF-alpha mRNA stability.

Blotting, Northern↗
PubMed1997

The von Hippel-Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity.

The von Hippel-Lindau tumor suppressor gene (VHL) has a critical role in the pathogenesis of clear-cell renal cell carcinoma (RCC), as VHL mutations have been found in both von Hippel-Lindau disease-associated and sporadic RCCs. Recent studies suggest that vascular endothelial growth factor (VEGF) mRNA is upregulated in RCC- and von Hippel-Lindau disease-associated tumors. We have therefore assessed the effect of the VHL gene product on VEGF expression. VEGF promoter-luciferase constructs were transiently cotransfected with a wild-type VHL (wt-VHL) vector in several cell lines, including 293 embryonic kidney and RCC cell lines. wt-VHL protein inhibited VEGF promoter activity in a dose-dependent manner up to 5- to 10-fold. Deletion analysis defined a 144-bp region of the VEGF promoter necessary for VHL repression. This VHL-responsive element is GC rich and specifically binds the transcription factor Sp1 in crude nuclear extracts. In Drosophila cells, cotransfected VHL represses Sp1-mediated activation but not basal activity of the VEGF promoter. We next demonstrated in coimmunoprecipitates that VHL and Sp1 were part of the same complex and, by using a glutathione-S-transferase-VHL fusion protein and purified Sp1, that VHL and Sp1 directly interact. Furthermore, endogenous VEGF mRNA levels were suppressed in permanent RCC cell lines expressing wt-VHL, and nuclear run-on studies indicated that VHL regulation of VEGF occurs at least partly at the transcriptional level. These observations support a new mechanism for VHL-mediated transcriptional repression via a direct inhibitory action on Sp1 and suggest that loss of Sp1 inhibition may be important in the pathogenesis of von Hippel-Lindau disease and RCC.

Animals↗
PubMed1996

Spectrum of mutations in the COL4A5 collagen gene in X-linked Alport syndrome.

Alport syndrome is a mainly X-linked hereditary disease of basement membranes that is characterized by progressive renal failure, deafness, and ocular lesions. It is associated with mutations of the COL4A5 gene located at Xq22 and encoding the alpha5 chain of type IV collagen. We have screened 48 of the 51 exons of the COL4A5 gene by SSCP analysis and have identified 64 mutations and 10 sequence variants among 131 unrelated Alport syndrome patients. This represents a mutation-detection rate of 50%. There were no hot-spot mutations and no recurrent mutations in our population. The identified mutations were 6 nonsense mutations, 12 frameshift mutations, 17 splice-site mutations, and 29 missense mutations, 27 of the latter being glycine substitutions in the collagenous domain. Two of these occurred on the same allele in one patient and segregated with the disease in the family. We showed that some of the glycine substitutions could be associated with the lack of immunological expression of the alpha3(IV)-alpha5(IV) collagen chains in the glomerular basement membrane.

Adolescent↗
PubMed1995

Autosomal recessive Alport syndrome: immunohistochemical study of type IV collagen chain distribution.

Alport syndrome (AS) is an hereditary disease of basement membrane collagen. It is mainly transmitted as a dominant X-linked trait and caused by mutations in the COL4A5 gene encoding the alpha 5 chain of type IV collagen. However, autosomal recessive AS due to mutations in the COL4A3 or COL4A4 genes could represent up to 15% of AS. Using the immunofluorescence technique, we analyzed the distribution of the different chains of type IV collagen in renal (12 specimens) and skin (4 specimens) basement membranes of 12 AS patients belonging to 11 unrelated kindreds in which autosomal recessive inheritance had been demonstrated (3 kindreds) or was suggested by clinical and genealogic data (8 kindreds). The renal and skin distribution was normal in one patient with COL4A4 mutations. A peculiar pattern of distribution of the alpha 3-alpha 5(IV) chains was observed in the other patients. It was characterized the co-absence of the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains in the glomerular basement membrane, and the presence of the alpha 5(IV) chain in a series of extraglomerular basement membranes including capsular, collecting ducts and epidermal basement membranes, a combination never observed in X-linked AS. This immunohistochemical pattern is correlated with the specific distribution of the alpha 3-alpha 5 chains of type IV collagen chains within extraglomerular basement membranes. It could be a useful marker for the identification of autosomal recessive AS.

Adolescent↗
PubMed1995

Splice-mediated insertion of an Alu sequence in the COL4A3 mRNA causing autosomal recessive Alport syndrome.

Alport syndrome is a mainly X-linked hereditary disease of basement membranes characterized by progressive renal failure, deafness, and ocular lesions. The alpha 3(IV) and alpha 4(IV) collagen genes have been recently shown to be involved in the less frequent autosomal recessive form. When screening lymphocyte COL4A3 mRNAs from Alport patients, we found a mutant whose transcripts were disrupted by a 74 bp insertion at the junction of exons IV or V and VI. The insertion derives from an antisense Alu element in COL4A3 intron V, which has been spliced into the alpha 3(IV) mRNA due to a G to T transversion activating a cryptic acceptor splice site in this Alu element. There is complete segregation of this mutation with the disease in the family. Our findings provide the first evidence for the pathogenic role of abnormal splicing of COL4A3. Moreover, we demonstrate the superiority of mutation screening at the mRNA level to detect a hitherto poorly recognized mutation mechanism in humans, splice-mediated insertion of an Alu fragment into a coding sequence.

Antisense Elements (Genetics)↗
PubMed1994

Aberrant splicing of the COL4A5 gene in patients with Alport syndrome.

A variety of mutations have been identified in the X-linked type IV collagen alpha 5 chain (COL4A5) gene in patients with Alport syndrome. A substantial number of these mutations were predicted to have an effect on RNA splicing. For 4 such mutations in our group of patients the effect of the DNA mutation on the COL4A5 mRNA structure and stability was analysed. An alteration of the invariant splice acceptor site of intron 41 resulted in a shift of the actual splicing to either a cryptic splice site within exon 42 or the normal splice site in the next intron. A single base substitution of the final nucleotide of exon 48 resulted in the removal of the entire exon. Two frameshift mutations, a 10 basepair duplication in exon 49 and a single base deletion in exon 41, were incorporated in the mRNA as such and resulted in a stretch of missense codons terminated by a premature stop codon. Exon skipping was occasionally observed in these samples, but not reproducibly in every experiment. In healthy controls exon skipping was never detected. Analysis of female carriers revealed that in only one case was the stability of the mutated mRNA reduced in comparison with the normal transcript. The extent to which the non-collagenous domain was predicted to be deleted correlated with the severeness of the disease.

Base Sequence↗
PubMed1994

Deletions in the COL4A5 collagen gene in X-linked Alport syndrome. Characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.

The type IV collagen alpha 5 chain (COL4A5) gene of 88 unrelated male patients with X-linked Alport syndrome was tested for major gene rearrangements by Southern blot analysis, using COL4A5 cDNA probes. 14 different deletions were detected, providing a 16% deletion rate in the COL4A5 gene in the patient population. The deletions are dispersed all over the gene with different sizes, ranging from 1 kb to the complete absence of the gene (> 250 kb) in one patient. In four patients with intragenic deletions, absence of the alpha 3 (IV) chain in the glomerular basement membrane was demonstrated by immunohistochemical studies. This finding supports the hypothesis that abnormalities in the alpha 5 (IV) chain may prevent normal incorporation of the alpha 3 (IV) chain into the glomerular basement membrane. Direct sequencing of cDNA amplified from lymphoblast mRNA of four patients with internal gene deletions, using appropriate combinations of primers amplifying across the predicted boundaries of the deletions, allowed us to determine the effect of the genomic rearrangements on the transcripts and, by inference, on the alpha 5 (IV) chain. Regardless of the extent of deletion and of the putative protein product, the 14 deletions occur in patients with juvenile-type Alport syndrome.

Base Sequence↗