Transforming growth factor-β1 (TGF-β1) regulates cell junction restructuring via Smad-mediated repression and clathrin-mediated endocytosis of nectin-like molecule 2 (Necl-2)

Abstract

Nectin-like molecule-2 (Necl-2), a junction molecule, is exclusively expressed by spermatogenic cells. It mediates homophilic interaction between germ cells and heterophilic interaction between Sertoli and germ cells. Knockout studies have shown that loss of Necl-2 causes male infertility, suggesting Necl-2-based cell adhesion is crucial for spermatogenesis. Transforming growth factor-βs (TGF-βs) are crucial for regulating cell junction restructuring that are required for spermatogenesis. In the present study, we aim to investigate the mechanism on how TGF-β1 regulates Necl-2 expression to achieve timely junction restructuring in the seminiferous epithelium during spermatogenesis. We have demonstrated that TGF-β1 reduces Necl-2 mRNA and protein levels at both transcriptional and post-translational levels. Using inhibitor and clathrin shRNA, we have revealed that TGF-β1 induces Necl-2 protein degradation via clathrin-dependent endocytosis. Endocytosis assays further confirmed that TGF-β1 accelerates the internalization of Necl-2 protein to cytosol. Immunofluorescence staining also revealed that TGF-β1 effectively removes Necl-2 from cell-cell interface. In addition, TGF-β1 reduces Necl-2 mRNA via down-regulating Necl-2 promoter activity. Mutational studies coupled with knockdown experiments have shown that TGF-β1-induced Necl-2 repression requires activation of Smad proteins. EMSA and ChIP assays further confirmed that TGF-β1 promotes the binding of Smad proteins onto MyoD and CCAATa motifs in vitro and in vivo. Taken together, TGF-β1 is a potent cytokine that provides an effective mechanism in controlling Necl-2 expression in the testis via Smad-dependent gene repression and clathrin-mediated endocytosis.

Figure 1. Effects of TGF-β1 on Necl-2 expression and localization in GC-1 spg cells.

Cells treated with vehicle (4 mM HCl/0.1%BSA) or TGF-β1 (5 ng/ml) were subjected for RT-PCR (A) and Real-time PCR (B) analysis using primer specific for Necl-2 gene. For RT-PCR, β-actin was co-amplified as an internal control. Necl-2 mRNA levels were normalized with β-actin. The authenticity of the PCR product was confirmed by sequencing analysis. For real-time PCR, GAPDH was used as an internal control for normalization. (C) Necl-2 protein level was analyzed by Western Blotting. (D) Immunofluorescence staining was performed in vehicle- and TGF-β1-treated cells. Cells were incubated with rabbit anti-Necl-2 antibody, followed by goat anti-rabbit IgG conjugated with FITC. Necl-2 appeared as green fluorescent. DAPI was used for nuclear staining. Scale bar  = 20 µm. Results of RT-PCR, real-time PCR and Western blotting are expressed as the mean±S.D. of three independent experiments. ns, not significant vs vehicle control; *, p<0.01 vs vehicle control; **, p<0.001 vs vehicle control.

Figure 2. Post-translational regulation of Necl-2 protein by TGF-β1 in GC-1 spg cells.

(A) Necl-2 protein stability was analyzed by cycloheximide assay. Cells were pre-treated with CHX (5 µg/ml, 30 min) before vehicle or TGF-β1 treatment. Western blotting was performed to determine Necl-2 protein level. To determine the potential post-translational pathway, cells were pre-treated with CHX, followed by (B) proteasome inhibitor MG132 for 30 min, (C) caveolin inhibitor nystatin for 30 min or (D) clathrin inhibitor CPZ for 1 h or 1.5 h prior to TGF-β1 treatment (24 h). Necl-2 protein level was then analyzed by Western Blotting. (E) Immunofluorescence staining was performed in cells pretreated with CPZ followed by vehicle or TGF-β1 treatment. Cells were incubated with rabbit anti-Necl-2 antibody, followed by goat anti-rabbit IgG conjugated with FITC. Necl-2 appeared as green fluorescent. DAPI was used for nuclear staining. Scale bar  = 20 µm. A–D, Results are expressed as the mean±S.D. of three independent experiments. ns, not significant vs vehicle control; *, p<0.01 vs vehicle control; **, p<0.001 vs vehicle control. CHX, cycloheximide; CPZ, chlorpromazine.

Figure 3. Effect of clathrin-shRNA on Necl-2 protein level and endocytosis assay.

(A) Cells were transfected with sh-vector, sh-ctrl or sh-clathrin (#1 and #2) (1 µg) for 48 h, subsequently treated with CHX, followed by vehicle or TGF-β1 treatment. Level of clathrin, Necl-2 and actin were analysed by Western blotting. Actin was used as an internal control. Necl-2 protein levels were normalized with actin. (B) Immunofluorescence staining was performed in cells transfected with sh-vector, sh-ctrl or sh-clathrin followed by vehicle or TGF-β1 treatment. Cells were incubated with rabbit anti-Necl-2 antibody, followed by goat anti-rabbit IgG conjugated with Alexa Fluor 555. Necl-2 appeared as red fluorescent. DAPI was used for nuclear staining. Scale bar  = 20 µm. (C) GC-1 spg cells were biotinylated for 30 min. Cells were then incubated with either vehicle or TGF-β1 at 35°C to allow endocytosis, and reactions were terminated at various time points. Total = total labeled cell surface protein without stripping. Cell surface protein without biotinylation served as the negative control. Biotinylated proteins were pulled down by UltraLink Immobilized NeutrAvidin Plus beads and were subjected to Western blotting. Equal amounts of proteins were used at each time points as assessed by Actin blot. Percentage of internalized and biotinylated proteins remaining in the cytosol in the presence of vehicle or TGF-β1 are shown. The percentage of internalized and biotinylated Necl-2 at time 0 was arbitrarily set at 100. Results are expressed as the mean ± S.D. of three independent experiments. ns, not significant vs corresponding vehicle control; **, p<0.001 vs corresponding vehicle control. CHX, cycloheximide; Veh, vehicle control.

Figure 4. Effect of TGF-β1 on Necl-2 mRNA stability and

promoter activity. (A) and (B) Analysis of Necl-2 mRNA stability was performed by actinomycin D (ActD) assay. Cells were pre-treated with ActD (5 µg/ml) for 2 h before vehicle or TGF-β1 treatment. RT-PCR (A) and real-time PCR (B) were performed to determine Necl-2 mRNA level. (C) Progressive 5′-deletion analysis of mouse Necl-2 promoter was performed between nt -502 and -1. A series 5′-deletion constructs and pEGFP vector were co-transfected into GC-1 spg cells followed by TGF-β1 treatment (5 ng/ml, 18 h). (D) Three putative cis-acting elements including MyoD, CCAATa and CCAATb motifs are located within the region between nt -159 and -1 (upper panel). Site-directed mutagenic constructs containing single, double or triple mutations and pEGFP vector were co-transfected into GC-1 spg cells followed by TGF-β1 treatment. pEGFP activity was used to normalize transfection efficiency. Promoter activity was represented as the fold change when compared with pGL-3 vector. (E) pGL-3 vector, p(-159/−1)Luc and pEGFP vector were co-transfected with si-Smad3 (#1/#2, 20 nM) or/and si-Smad4 (#1/#2, 20 nM) for 48 h followed by TGF-β1 treatment. pEGFP activity was used to normalize transfection efficiency. Smad3 and Smad4 protein levels were examined by Western blotting. (F) Wild-type and single mutated constructs of p(-159/−1)Luc were co-transfected with pcDNA3.1 vector, Smad3 or/and Smad4 expression constructs into GC-1 spg cells. The promoter activity was presented as a percentage of that of pcDNA3.1-transfected cells. (A–F), Results are expressed as the mean±S.D. of three independent experiments. ns, not significant vs vehicle control (A–E) or p(-159/−1)Luc (F); *, p<0.01 vehicle control (A–E) or vs p(-159/−1)Luc (F); **, p<0.001 vs vehicle control (A–E) or p(-159/−1)Luc (F). ActD, actinomycin D.

Figure 5. EMSA of MyoD and CCAATa motifs and ChIP assay.

(A) and (D) Dose-dependent and competition assay for EMSA of MyoD motif (A) and CCAATa motif (D). Double stranded oligonucleotides containing the respective motif were radiolabeled with [γ-³²P]ATP and incubated with nuclear extract (1–15 µg) alone or in the presence of cold competitors (100- to 500-fold excess). (B–C) and (E–F) Labeled probes were incubated with vehicle or TGF-β1 treated nuclear extract (15 µg for MyoD motif and 10 µg for CCAATa motif) in the presence of specified antibodies or rabbit serum (Rb serum). (G) A schematic drawing illustrating the relative location of MyoD and CCAAT cis-acting motifs in the Necl-2 promoter and chromatin immunoprecipitation assay. TGF-β1-treated genomic DNA-protein samples were immunopreciptated with antibodies against Smad3 and Smad4 (2 µg) or rabbit serum. Precipitated DNA-protein complexes were subjected to DNA purification. The promoter region and the open reading frame of Necl-2 gene were amplified using specific primer pairs no. A287/A288 (for promoter region) and no. A107/A108 (for open reading frame), respectively, followed by agarose gel electrophoresis. Rb, Rabbit. Cpx, complex.