Alternative splicing of TGF-betas and their high-affinity receptors T beta RI, T beta RII and T beta RIII (betaglycan) reveal new variants in human prostatic cells

Abstract

Background: The transforming growth factors (TGF)-beta, TGF-beta1, TGF-beta2 and TGF-beta 3, and their receptors [T beta RI, T beta RII, T beta R III (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 T beta RIB, T beta RIIB and TGF-beta 2B in human prostatic cells.

Results: Interestingly, a novel human receptor transcript T beta RIIC 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 T beta RIB with four additional amino acids was identified also in human. Expression of the variant T beta RIIB 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 T beta RIIC and TGF-beta 2B mainly in the epithelial cells with a preferential localization of TGF-beta 2B in the apical cell compartment. Whereas in the androgen-independent hPCPs and PC-3 cells all TGF-beta ligands and receptors are expressed, the androgen-dependent LNCaP cells failed to express all ligands. Additionally, stimulation of PC-3 cells with TGF-beta2 resulted in a significant and strong increase in secretion of plasminogen activator inhibitor-1 (PAI-1) with a major participation of T beta RII.

Conclusion: In general, expression of the splice variants was more heterogeneous in contrast to the well-known isoforms. The identification of the splice variants T beta RIB and the novel isoform T beta RIIC in man clearly contributes to the growing complexity of the TGF-beta family.

Figure 1

(A) Comparison of the exon structure of the human TβRI mRNA with the pseudogene on chromosome 19. 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. (B) 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. (C) 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.

Figure 2

(A) 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. (B) 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. (C) 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).

Figure 3

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

Figure 4

(A) Comparison of the exon structure of the human TβRII mRNA with the truncated sequence provided by Yang et al. [35]. Lines depict the 5'-UTR, 3'-UTR and ESTs with additional exons. (B) 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. (C) 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.

Figure 5

(A) Exon structure of the human TβRIII (betaglycan) mRNA. Lines depict the 5'-UTR and 3'-UTR. Expression pattern of the TβRIII gene in human prostatic cells (5-HTBR3E13/3-HTBR3E15). (B) Exon structure of the human TGF-β1 mRNA. Lines depict the 5'-UTR and 3'-UTR. Expression pattern of the TGF-β1 gene in human prostatic cells (5-HTGFB1E3/3-HTGFB1E6). Ctrl, control.

Figure 6

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

Figure 7

(A) Comparison of the exon structure of the human TGF-β2 mRNA with the ESTs BP214137 and BF752669 containing the additional alternative exon 2B. Lines depict the 5'-UTR, 3'-UTR and introns. (B) 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. (C) Exon structure of the human TGF-β3 mRNA. Lines depict the 5'-UTR and 3'-UTR. (D) 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.

Figure 8

(A) Localization of TβRIIB in human prostate carcinoma is found in most epithelial cells (mainly in basal but also in columnar cells) in a nontumorous gland adjacent to a nontumorous gland without staining. (B) The negative control did not reveal any staining. Localization of TβRIIC in human prostate carcinoma was found in epithelial cells (C) and muscle cells (D). TGF-β2B was localized primarily in the apical region of epithelial cells (E). (F) The negative control did not reveal any staining. A-F, 100× magnification

Figure 9

Secretion of PAI-1 by PC-3 cells was quantified by ELISAs. TGF-β2 alone (-) stimulated secretion of PAI-1 significantly compared to the control (Ctrl). Antibody perturbation experiments with antibodies specific for the extracellular domains of TβRII (RII), the alternative exons of TβRIIB (RIIB) and TβRIIC (RIIC), demonstrated a significant decrease in the amount of PAI-1 only for TβRII compared to the stimulation with TGF-β2 (-) or the unspecific antibody (ctrl-R). For the sake of clarity, we have not indicated that the antibody perturbation experiments were also significantly different to the control (Ctrl) without any TGF-β2 treatment. An unspecific antibody (ctrl-R) did not inhibit PAI-1 secretion stimulated by TGF-β2. Each experiment was independently repeated five times (n = 5) in duplicate, with each value given as the mean ± SEM. Statistically significant differences are indicated (*, P < 0.05; ***, P < 0.001).