Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells
© Konrad et al; licensee BioMed Central Ltd. 2007
Received: 24 May 2007
Accepted: 11 September 2007
Published: 11 September 2007
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.
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.
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.
Transforming growth factor (TGF)-β is a secreted cytokine implicated in a wide variety of biological processes such as apoptosis, motility, tumorigenesis, proliferation, differentiation, and gene expression . In mammals three TGF-betas, TGF-β1, TGF-β2 and TGF-β3, have been cloned, and although they show very often overlapping functions in vitro, the isoform-specific knockout mice revealed non-redundant and non-overlapping phenotypes. Approximately 50% of the TGF-β1 knockout mice died during embryogenesis from yolk sac defects; the survivors developed inflammatory disorders and died typically within one month [2–4]. TGF-β2 knockout mice were perinatally lethal and developed defects in different organs such as heart, kidney, testis, as well as various craniofacial defects and axial and appendicular skeletal defects . Moreover, TGF-β3 knockout mice were perinatally lethal due to a delayed lung development and defective palatogenesis [6, 7].
Secretion and inactivation of the TGF-betas is regulated by the latency-associated peptides (LAPs) which are generated from the respective N-terminal TGF-β proteins by cleavage . Additionally, latent TGF-β binding proteins (LTBP1-4) are covalently attached to the LAPs and mediate storage in the extracellular matrix (ECM). After activation by proteolytic enzymes or acidic environment the TGF-betas bind with high affinity to the serine/threonine kinase receptor TβRII which in turns phosphorylates TβRI. Activation of the receptor complex propagates the signal downstream to the Smad proteins, who regulate many TGF-β-induced transcriptional responses . Alternatively, TGF-β2 can bind to the accessory receptor TβRIII, who facilitates binding of TGF-β2 to TβRII. However, signal transduction is initiated again by TβRI. Recently, it was shown that TGF-β2 might also bind to an alternative splice product of the TβRII gene, mainly expressed in osteoblasts and mesenchymal cells. The receptor isoform TβRIIB binds TGF-β2 also in the absence of TβRIII and then activates TβRI starting the signal transduction . However, recently it was shown in human chondrocytes that TβRIIB exerted a higher affinity for TGF-β1 than for TGF-β2 . In addition to alternative splicing of TβRII, TGF-β2 and TβRI also were demonstrated to be alternatively spliced in human prostatic PC-3 cells  and embryonic stem cells from mouse , respectively.
TGF-betas are believed to be involved in several aspects of carcinogenesis. At the beginning of tumor formation the TGF-betas might inhibit proliferation of cancer cells, but with ongoing dedifferentiation, the TGF-betas and the receptors seem to enhance motility and metastasis of the tumor cells [1, 14]. In more advanced and especially metastasised stages higher serum levels of TGF-β1 were found  and reduced expression of TβRII and TβRI in the tumor tissue was associated with poor prognosis .
Recently, analyses of alternative splicing indicated that approximately 40–60% of human genes express splice variants, suggesting that alternative splicing contributes to the growing complexity of the human genome . In many transcripts, addition or deletion of complete exons or introduction of an early stop codon may result in a truncated or unstable mRNA . Alternative splicing has been shown to be involved in ligand binding to growth factor receptors like TβRIIB , cell adhesion or various human diseases . Additionally, alternative splicing occurs sometimes during developmental processes and may be restricted to distinct tissues . Interestingly, it was reported that more alternative splicing was found in organs such as testis, pancreas, placenta, and liver . Up to date many groups have presented genomic analyses of alternative splicing by use of expressed sequence tags (EST, [e.g. [21–23]] or microarrays . Most of these results are now available in databases .
In this study, we have analyzed the mRNA expression of the TGF-betas and the receptors TβRI-III mainly in human prostatic cells available to us and identified the splice variants TGF-β2B, TβRIB, TβRIIB and the new variant TβRIIC. Of note, the alternatively spliced exons were found in the N-terminal part of the proteins and extracellular domains of the receptors. The splice variant TGF-β2B could be identified in more species than the other isoforms and showed less sequence variation among the various species. Furthermore, this is the first report showing localization of the splice variants TβRIIB, TβRIIC and TGF-β2B in human prostate tissue.
Literature and database search for alternative splicing
The search in the literature (PubMed) and sequence databases for TGF-betas and their high-affinity receptors displayed deleted or additional exons. Alternative splicing of the TGF-beta ligands was described for TGF-β1 in pig  and for TGF-β2 in human and rat [12, 25]. Alternative splicing of the high-affinity receptors was demonstrated for TβRI in mouse, rat and boar [13, 26, 27], and for TβRII in mouse and human [28–30].
In the database ASDB , dealing with alternative splicing, TGF-β2 and TGF-β3 were mentioned to contain splice variants, and in the database ASAP  three isoforms for TGF-β1 were described. The database EASED  showed many but not all of the aberrant ESTs which were found in this study.
Alternative splicing and mRNA expression of TβRI
Primer pairs used for characterization
Gene (Acc No)a
Alternative splicing and mRNA expression of TβRII
Alternative splicing and mRNA expression of TβRIII (betaglycan)
Alternative splicing and mRNA expression of TGF-β1
The alignment of the TGF-β1 gene with the ESTs did not show any new exons. Because in the TGF-β2 gene the alternatively spliced exon 2B between the first two exons was found as mentioned above, we tested whether this was also the case for the TGF-β1 cDNA. However, in the prostatic cells no additional exon was identified (data not shown). Besides LNCaP all cell lines studied showed expression of TGF-β1 (Fig. 5B). Additionally, we tested whether exons 4 and 5 were deleted in the human sequence as has been published for the porcine sequence . However, in the prostatic cell lines studied this deletion was not detectable (Fig. 5B).
Alternative splicing and mRNA expression of TGF-β2
Alternative splicing and mRNA expression of TGF-β3
The alignment of the TGF-β3 cDNA sequence with the EST database only yielded incorrectly spliced exons (Fig. 7C). We found an annotation for alternative splicing of TGF-β3 in the ASDB database . Although the TGF-β3 gene could be found in this genomic clone, the alternative splicing does belong to the next gene, adjacent to TGF-β3. mRNA expression of TGF-β3 was investigated with primers located in exon 1 and exon 2 to test for possible new exons. However, we only observed one specific amplicon in all prostatic cell lines except for LNCaP cells (Fig. 7D). Expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for all cell lines used is shown in Fig. 7D.
Localization of the alternative splice variants
TβRIIC was localized in the epithelial cells (Fig. 8C) in very few glands of the human prostate (histological grading pT2apN0M0) and also in muscle cells (Fig. 8D, histological grading pT3bpN0M0). The splice variant TGF-β2B was found also mainly in the epithelial cells in the apical region (Fig. 8E, histological grading pT2bpN0M0). The negative control without the primary antibody did not show any staining (Fig. 8F).
Functional analysis of the alternative splice variants
Expression pattern of the TGF-β ligands, receptors and splice variants
mRNA Expression of prostatic TGF-betas and their receptors
Analysis of mRNA expression of TGF-betas and their receptors in human prostatic cell lines showed very controversial results. It is generally accepted that PC-3 cells express TGF-β1, TGF-β2 and TGF-β3 [37, 38] and even more the TGF-β2 gene was originally cloned from PC-3 cells . In line with this, our study also showed expression of all TGF-beta ligands in the stromal hPCPs and PC-3. However, experiments with LNCaP yielded controversial results. Whereas mRNA expression for TGF-β1 to TGF-β3 was demonstrated  all other studies including this one could not find mRNA expression of TGF-β1 in LNCaP cells [39, 40]. Our study showed expression of all TGF-beta ligands in the stromal cells hPCPs which is in accordance with the results for other stromal cell lines derived from the human prostate . However, this study confirmed an earlier report showing that PC-3 cells express the splice variant TGF-β2B .
PC-3 cells showed expression of TβRI and TβRII, which was confirmed in this study. In LNCaP cells expression of TβRII was found, but TβRI was not expressed [42, 43]. However, another study  like the present detected mRNA expression by RT-PCR in LNCaP cells. For stromal prostatic cells expression of TβRI and TβRII was found by us and others . This is the first study to show expression of TβRIII and the receptor splice variants TβRIB and TβRIIB and to identify a novel transcript termed TβRIIC which will be discussed below. Only the stromal cells hPCPs expressed all receptor splice variants as well as TGF-β2B (Table 2). Except for the splice variant TβRIB, PC-3 cells also expressed all receptor splice variants, whereas LNCaP cells did not express TGF-β2B which is in line with the missing expression of all TGF-β ligands. Interestingly, these cells did also not express the newly identified splice variant TβRIIC (Table 2).
Localization and protein data of the splice variants of TGF-β ligands and receptors
Agrotis et al.  have demonstrated, that TβRI is more abundant in contractile smooth muscle cells than the variant TβRIB. Additionally, they found that TβRI displayed a greater ability to induce PAI-1 mRNA in response to TGF-β1, whereas TβRIB performed slightly better in growth inhibition . Interestingly, we identified for TβRI a pseudogene on chromosome 19, reaching from exon 2 to exon 4 and a short stretch of 62 bp from the 3'-UTR of the gene.
In contrast to TβRI which mainly is important for signal transduction, TβRII is involved in direct interaction with the ligands TGF-β1, TGF-β2 and TGF-β3 . In TβRII the additional exon 2B was hypothesized to be involved in high-affinity binding of TGF-β2 to the receptor isoform TβRIIB also in the absence of TβRIII . However, it was shown recently that TGF-β2 could bind to soluble TβRIIB or TβRII only in combination with soluble TβRI  and that also TGF-β1 could interact with TβRIIB . Furthermore, the TβR knockouts, TβRI , TβRII  and TβRIII , revealed non-overlapping phenotypes with the TGF-β2 null mice , although TβRIII knockouts displayed reduced TGF-β2 binding . This implies that either the high-affinity receptor for TGF-β2 is still not found or that receptor combinations might be responsible for the interaction.
Expression of TβRII was found in the human prostate in normal and tumor tissue primarily in the epithelial cells with a diminished expression in more advanced stages . Similarily our results with TβRIIB also showed a distinct localization in the epithelial cells of normal and tumor tissue of the human prostate.
Our analysis clearly showed the expression of a novel transcript variant TβRIIC in PC-3, hPCPs cells, Caco-2 and up to 20 normal tissues including human prostate, indicating a ubiquitous expression in human organs. The additional and alternatively spliced exon encodes 36 amino acids located in the extracellular domain in close proximity to the transmembrane domain. Although the database search for protein domains revealed no similarities to other proteins or specific motifs, it is noteworthy, that the additional domain contains two additional cysteines which might be important for protein folding. Interestingly, we found a deletion of 4 bp at the 5'-end of the additional exon 4B in TβRIICΔ4, possibly resulting in a truncated receptor. Although expressed at a very low level, it was found in normal tissue and preliminary results suggest this to be also the case in tumor samples. Interestingly protein localization of TβRIIC was also found mainly in the epithelial cells of the human prostate but in very few glands.
The splice variant TGF-β2B mRNA was first described in the prostatic cell line PC-3 [12, 50] and in rats in skeletal muscles, aorta, primary bronchus, heart, uterus, sciatic nerve, and spinal cord . Additionally, TGF-β2B mRNA and protein were found in most somatic and germinal cells of mouse and rat . TGF-β2B was also demonstrated to be secreted by BSC-40 cells from monkeys . The additional exon of TGF-β2B is part of the LAP-domain which is important for correct secretion and inactivation of the mature C-terminal TGF-β2 dimer . The alternatively spliced exon 2B contains 3 additional cysteine residues which might be important for the formation of cysteine bonds and therefore might influence protein folding. However, TGF-β2B is secreted and forms a latent complex with the LAP . It is important to note that TGF-β2B is cleaved similarly to TGF-β2 and yields a mature monomer/dimer of exactly the same size as mature TGF-β2 . Because only mature TGF-β2 binds to the receptor it is equal whether mature TGF-β2 is cleaved from the short TGF-β2 variant or long TGF-β2B variant. Whether the existence of the two different TGF-β2 LAP complexes is required for different binding to LTBPs and thus might be stored differently in the ECM warrants further investigation.
Up to date TGF-β2B was identified in most species, whereas TβRIB and TβRIIB were found in fewer species. It is noteworthy that TβRIIB is not as well conserved between human and mouse than TGF-β2B and up to date was not found in rat . Therefore, we conclude that TβRIIB is not as ubiquitously expressed in the different species like the other variants and therefore could not serve as a ubiquitous receptor for TGF-β2. In line with this assumption, we could observe only a moderate decrease in PAI-1 secretion after inhibition of TβRIIB or TβRIIC after stimulation of PC-3 cells with TGF-β2. However, this is the first report showing a 10-fold increase of PAI-1 secretion in PC-3 cells after stimulation with TGF-β2.
In general, mRNA expression of the TGF-β and TβR splice variants was more heterogeneous and weaker compared to the variants without the alternative exons. The variant TGF-β2B was identified in most species and is up to date the best conserved isoform among the various species. Similarly, the splice variant TβRIB was also found in many species in contrast to the isoforms TβRIIB and TβRIIC which showed a more restricted species distribution. This is the first report showing a distinct localization of TGF-β2B, TβRIIB and TβRIIC in the human prostate mainly in the epithelium.
Cell lines and tissues
The stromal cells hPCPs from the human prostate were propagated as described . LNCaP and PC-3 cells were purchased from American Type Culture Collection (ATCC) and cultivated as published . Colon cancer cell line Caco-2 was purchased from ATCC and kindly provided by Dr W.W. Franke (German Cancer Research Center, Heidelberg, Germany) and kept under standard conditions. Total RNA from 20 normal human tissues was purchased (Becton Dickinson, Heidelberg, Germany).
RNA isolation, cDNA synthesis and RT-PCR
Total RNA from the cell lines was isolated with Trizol (Gibco BRL, Karlsruhe, Germany) according to the manufacturer's instructions. Total RNA of Caco-2 cells was isolated using RNAeasy isolation kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. Reverse transcription was performed using 2 μg of total RNA and Omniscript (Qiagen), except for total RNA from Caco-2, which was reverse transcribed as described elsewhere . Primers used for PCR are denoted in Table 1 and were intron-spanning to overcome genomic contamination. PCR was performed on a Hybaid Omnigene Thermocycler (MWG Biotech, Ebersberg, Germany) using mainly PanScript Taq polymerase (Pansystems, Aidenbach, Germany) as described . Amplification with the primers 5-TGFB3E1/3-TGFB3E2 was performed with the Platinum Taq Polymerase (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. The first round of the nested PCR to clone TβRIIC was done with the primers 5-HTBR2E3/3-HTBR2E4 from which 20 μl were used for the second round with the primers 5-HTBR2Z/3-HTBR2E4. The other fragment of Tβ RIIC was also cloned after a nested PCR with the primers 5-HTBR2B/3-HTBR2CD in the first round and primers 5-HTBR2E3/3-HTBR2CD were used for the second round. The nested PCR was performed with the Qiagen Taq DNA Polymerase and solution Q (Qiagen) on a PTC100 cycler (Biozym, Germany). Amplification was carried out for 35 cycles, except for 5-GAPDH/3-GAPDH which was run for 25 cycles and 5-HTBR2E3/HTBR2CD which was run for 30 cycles. After an initial heating to 94°C for 4 min, each cycle consisted of denaturing at 94°C for 45 sec, annealing at the temperatures indicated in Table 1 for 45 sec and elongation at 72°C for 90 sec except for the last extension which lasted 5 min. PCR products were separated on agarose gels, extracted with Qiaex (Qiagen), subcloned into the pCR2.0 vector (Invitrogen) and subsequently sequenced by MWG Biotech and GENterprise (Mainz, Germany). Amplification with the Cy-5 labeled primer 5-HTBR2E3 with the primer 3-HTBR2CD to detect TβRIICΔ4 was performed as described , except that cDNA instead of genomic DNA was used. PCR fragments were separated on 8% polyacrylamide gels .
Screening for alternatively spliced ESTs
The exon and intron pattern of the TGF-betas and their receptors was either found in the NCBI sequence database or determined by sequence comparison of the cDNAs with the genomic sequences by using the Blast tool. Each exon of the respective cDNAs was aligned with all available ESTs from human. Then, every EST was aligned with the genomic sequences to find alternatively/incorrectly spliced exons, which were analyzed for standard splice sites (GT-AG at the 5'- and 3'-end, respectively) and for a continuous open reading frame. Only good candidates which fulfilled both criteria were further analyzed by RT-PCR.
Generation of polyclonal antibodies
Polyclonal antibodies directed against the peptide SFCSIQSQVLCGYLD of the alternative exon of the rat TGF-β2B (Fig. 6) and against the peptide IRHIGSNNRLQRSTC of the alternative exon of TβRIIC (Fig. 3) were raised in two rabbits respectively according to standard protocols (Coring, Gernsheim, Gemany) as published . These peptide sequences are highly homologous in most species and did not show any homology to other proteins. Polyclonal antibodies were also affinity-purified on a sepharose column. Specificity of the antibodies was tested in ELISAs (CovAbtest, Coring) and western blots. Negative controls were performed with the preimmune serum and showed no binding.
Analysis of localization of TβRIIB, TβRIIC and TGF-β2B
Polyclonal antibody against TβRIIB was purchased from R&D Systems (Wiesbaden, Germany) and diluted 1:50 for immunohistochemistry. Polyclonal antisera against TβRIIC and TGF-β2B were used at dilutions of 1:50 and 1:100. Negative controls were performed by omitting the primary antibodies. Immunohistochemistry was done with the Envision System from DAKO (Hamburg, Germany) according to the instructions of the manufacturer with DAB staining and HE counterstaining.
PAI-1 ELISA and antibody perturbation
Quantitation of PAI-1 was performed with the highly sensitive PAI-1 Antigen ELISA Kit (Technoclone, Vienna, Austria), according to the manufacturer's instructions. PC-3 cells (50,000 cells/well) were seeded on 24-well plates and grown in DMEM (+10% FCS and antibiotics) at 37°C and 5% CO2 for 24 h. Then, medium was changed to DMEM containing the antibodies against TβRII (diluted 1:12.5, AF-241-NA, R&D Systems), TβRIIB (diluted 1:12.5, AF1300, R&D Systems), and TβRIIC (diluted 1:12.5). Control incubations were performed (i) without antibody, and (ii) by replacement of the antibodies by anti-goat IgG (1:12.5; Invitrogen, Karlsruhe, Germany). After incubation at 37°C and 5% CO2 for 1 h, TGF-β2 (10 ng/ml) was added. The cells were grown at 37°C for 72 h and collected supernatants were stored with protease-inhibitors (Complete Mini, Roche, Mannheim, Germany) at -20°C until the PAI-1 ELISA was performed.
All experiments were repeated independently at least three times in duplicate. Values from all experiments were used for calculation of the means and their respective standard errors of the mean (SEM). Statistical tests of one way analysis of variance (ANOVA) followed by the non-parametric test of Kruskal Wallis were used to determine significant differences between different experimental groups and the controls by using GraphPad Instat 3 (GraphPad, San Diego, USA). P values less than 0.05 were considered statistically significant.
- Acc No:
EMBL/DDBJ/GenBank Accession Number
analysis of variance
American Type Culture Collection
expressed sequence tag
Ser/Thr kinase domain
latent TGF-β binding protein
plasminogen activator inhibitor-1
standard error of the mean
transforming growth factor-beta
This study was financed by the Deutsche Forschungsgemeinschaft GK533 and the Kulemann-Stiftung of the University of Marburg. We thank Dr GH Lüers for insightful comments during the preparation of the manuscript and H Kirchner, R Nottelmann and B Lauer for technical assistance.
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