Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells

<p>Abstract</p> <p>Background</p> <p>The transforming growth factors (TGF)-β, TGF-β1, TGF-β2 and TGF-β3, and their receptors [TβRI, TβRII, TβRIII (betaglycan)] elicit pleiotropic functions in the prostate. Although expression of the ligands and receptors have been investigated, the splice variants have never been analyzed. We therefore have analyzed all ligands, the receptors and the splice variants TβRIB, TβRIIB and TGF-β2B in human prostatic cells.</p> <p>Results</p> <p>Interestingly, a novel human receptor transcript TβRIIC was identified, encoding additional 36 amino acids in the extracellular domain, that is expressed in the prostatic cancer cells PC-3, stromal hPCPs, and other human tissues. Furthermore, the receptor variant TβRIB with four additional amino acids was identified also in human. Expression of the variant TβRIIB was found in all prostate cell lines studied with a preferential localization in epithelial cells in some human prostatic glands. Similarly, we observed localization of TβRIIC and TGF-β2B mainly in the epithelial cells with a preferential localization of TGF-β2B in the apical cell compartment. Whereas in the androgen-independent hPCPs and PC-3 cells all TGF-β ligands and receptors are expressed, the androgen-dependent LNCaP cells failed to express all ligands. Additionally, stimulation of PC-3 cells with TGF-β2 resulted in a significant and strong increase in secretion of plasminogen activator inhibitor-1 (PAI-1) with a major participation of TβRII.</p> <p>Conclusion</p> <p>In general, expression of the splice variants was more heterogeneous in contrast to the well-known isoforms. The identification of the splice variants TβRIB and the novel isoform TβRIIC in man clearly contributes to the growing complexity of the TGF-β family.</p

Metabolic biomarker signature to differentiate pancreatic ductal adenocarcinoma from chronic pancreatitis

Objective Current non-invasive diagnostic tests can distinguish between pancreatic cancer (pancreatic ductal adenocarcinoma (PDAC)) and chronic pancreatitis (CP) in only about two thirds of patients. We have searched for blood-derived metabolite biomarkers for this diagnostic purpose. Design For a case-control study in three tertiary referral centres, 914 subjects were prospectively recruited with PDAC (n=271), CP (n=282), liver cirrhosis (n=100) or healthy as well as non-pancreatic disease controls (n=261) in three consecutive studies. Metabolomic profiles of plasma and serum samples were generated from 477 metabolites identified by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Results A biomarker signature (nine metabolites and additionally CA19-9) was identified for the differential diagnosis between PDAC and CP. The biomarker signature distinguished PDAC from CP in the training set with an area under the curve (AUC) of 0.96 (95% CI 0.93-0.98). The biomarker signature cut-off of 0.384 at 85% fixed specificity showed a sensitivity of 94.9% (95% CI 87.0%-97.0%). In the test set, an AUC of 0.94 (95% CI 0.91-0.97) and, using the same cut-off, a sensitivity of 89.9% (95% CI 81.0%-95.5%) and a specificity of 91.3% (95% CI 82.8%-96.4%) were achieved, successfully validating the biomarker signature. Conclusions In patients with CP with an increased risk for pancreatic cancer (cumulative incidence 1.95%), the performance of this biomarker signature results in a negative predictive value of 99.9% (95% CI 99.7%-99.9%) (training set) and 99.8% (95% CI 99.6%-99.9%) (test set). In one third of our patients, the clinical use of this biomarker signature would have improved diagnosis and treatment stratification in comparison to CA19-9

() Comparison of the exon structure of the human TβRII mRNA with the truncated sequence provided by Yang et al

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> [35]. Lines depict the 5'-UTR, 3'-UTR and ESTs with additional exons. () Expression pattern of both transcript variants of the TβRII gene in human prostatic cells (upper panel, 5-HTBR2B/3-HTBR2B). Expression of the novel splice variant TβRIIC in human prostatic cells (lower panel, nested PCR first round 5-HTBR2E3/3-HTBR2E4, second round 5-HTBR2Z/3-HTBR2E4) and normal human tissues (5-HTBR2E3/3-HTBR2CD) is shown. Additionally, GAPDH expression is also provided. () Fluorescence detection of TβRIICΔ4 (5-HTBR2E3/3-HTBR2CD, arrows) and TβRIIC is demonstrated. Caco, Caco-2; ctrl, control; g, gland; m, muscle; mu, mucosa; s, small; ma, marrow

() Schematic drawing of the TβRII protein (EC, extracellular domain; TM, transmembrane domain; Kinase, Ser/Thr kinase domain) with the two alternatively spliced exons 2B and 4B

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> () Nucleotide sequence of the cDNA and deduced amino acid sequence of exon 2B (underlined capital letters) and splice site junctions (lower case letters) of the variant TβRIIB are shown. () Additionally, the partial nucleotide and amino acid sequence of TβRII without exon 2B is shown. Underlined amino acids indicate amino acid exchange at the splice site junction due to the alternative splicing. Bold letters mark the amino acid and nucleotide exchanges with respect to the human sequence. Arrows indicate the exon boundaries. (hs, homo sapiens; pt, pan troglodytes; mmu, macaca mulatta; mm, mus musculus)

Schematic drawing of the TGF-β2 protein (LAP, latency-associated peptide) with the alternatively spliced exon 2B

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> Nucleotide and amino acid sequence of exon 2B (underlined capital letters) of the variant TGF-β2B are shown. Additionally, the partial sequence of TGF-β2 without exon 2B is shown. The sequence of TGF-β2 was not available for oryctolagus cuniculus. Underlined amino acids indicate amino acid exchange at the splice site junction due to the alternative splicing. Bold letters mark the amino acid and nucleotide exchanges with respect to the human sequence. Arrows indicate the exon boundaries. The accession numbers are also given. (hs, homo sapiens; pt, pan troglodytes; mmu, macaca mulatta; cf, canis familiaris; oc, oryctolagus cuniculus; mm, mus musculus; rn, rattus norvegicus)

Nucleotide and amino acid sequence of exon 4B (underlined capital letters) of the variant TβRIIC and TβRIICΔ4 are given

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> Furthermore, the partial nucleotide and amino acid sequences of TβRII without exon 4B are shown. Bold letters mark the amino acid and nucleotide exchanges with respect to the human sequence. Arrows indicate the exon boundaries. (hs, homo sapiens; pt, pan troglodytes; mmu, macaca mulatta)

() Comparison of the exon structure of the human TGF-β2 mRNA with the ESTs BP214137 and BF752669 containing the additional alternative exon 2B

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> Lines depict the 5'-UTR, 3'-UTR and introns. () Expression of both transcript variants (upper panel, 5-TGFB2E1B/3-TGFB2E2B) and expression of the splice variant TGF-β2B (lower panel; 5-HTB2CP/3-HTB2CP) is shown. () Exon structure of the human TGF-β3 mRNA. Lines depict the 5'-UTR and 3'-UTR. () Expression pattern of the TGF-β3 gene in human prostatic cells (left panel, 5-TGFB3E1/3-TGFB3E2). Additionally, GAPDH expression of all cell lines studied is shown. Ctrl, control

() Comparison of the exon structure of the human TβRI mRNA with the pseudogene on chromosome 19

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> A detailed alignment of the pseudogene with exons 2 to 4 and the 3'-UTR is available from the authors upon request. Lines depict the 5'-UTR and 3'-UTR. The repetitive elements AluSX, AluSB and L1Pa13 are encircled. () Expression pattern of the TβRI gene in human prostatic cells. Expression of both transcript variants (upper panel, 5-TB1RL/3-TB1RL) and expression of the splice variant TβRIB (lower panel; 5-TB1RL/3-HTB1RL) is demonstrated. () Scheme of the TβRI protein (EC, extracellular domain; TM, transmembrane domain; kinase, Ser/Thr kinase domain) with the nucleotide and amino acid sequence of exon 2 and exon 3 (capital letters) and the alternatively spliced exon 3B (lower case letters). Additionally, the partial sequence without the alternatively spliced exon is given below. The sequence of TβRI was not available for canis familiaris. The splice site junctions are indicated by italic letters. Bold letters mark the amino acid and nucleotide exchanges with respect to the human sequence. The accession numbers are given below (hs, homo sapiens; mm, mus musculus; rn, rattus norvegicus; ss, sus scrofa; cf, canis familiaris). Arrows indicate the exon boundaries. Ctrl, control