Transglutaminase-1 regulates renal epithelial cell proliferation through activation of Stat-3

Abstract

Transglutaminase-1 (TGase-1) is a Ca(2+)-dependent enzyme capable of cross-linking a variety of proteins and promoting wound healing in the skin. In this study, we examined the role of TGase-1 in proliferation of renal proximal tubular cells (RPTC). TGase-1, but not TGase-2, -5, and -7, was expressed in RPTC. Treatment with monodansylcadarevine (MDC), a selective TGase inhibitor or down-regulation of TGase-1 with small interfering RNA (siRNA) decreased RPTC proliferation. Proliferation of RPTC was accompanied by activation of Akt and Stat-3 (signal transducer and activator of transcription-3). Treatment with MDC or TGase-1 siRNA decreased Stat-3 but not Akt phosphorylation. Further studies showed that the Janus-activated kinase 2 (JAK2) mediates phosphorylation of Stat-3, and knockdown of either JAK2 or Stat-3 by siRNA decreased RPTC proliferation. However, inhibition of TGase-1 decreased phosphorylation of Stat-3 but not JAK2. Overexpression of Stat-3, JAK2, and/or TGase-1 in RPTC revealed that JAK2 is indispensable for TGase-1 to induce Stat-3 phosphorylation and TGase-1 potentiates JAK2-induced Stat-3 phosphorylation. Consistent with these observations, we found that inhibition of TGase-1 and the JAK2-Stat-3 signaling pathway decreased the transcriptional activity of Stat-3 and expression of the Stat-3-targeted genes, cyclin D1 and cyclin E. Conversely, overexpresssion of TGase-1 enhanced the JAK2-dependent transcriptional activity of Stat-3. Finally, TGase-1 was found to interact with JAK2, and this interaction was inhibited by MDC. These results demonstrate that TGase-1 plays an important role in regulation of renal epithelial cell proliferation through the JAK2-Stat-3 signaling pathway.

FIGURE 1.

Concentration-dependent inhibition of cell proliferation by MDC in cultured RPTC. RPTC were cultured for 24 h in the presence of 5% FBS and then treated with MDC for an additional 48 h at the indicated concentrations (A and B). Cell proliferation was assessed by the MTT assay (A) or by counting cell numbers (B). RPTC were starved for 24 h and then incubated in the presence of 5% FBS with 100 μm MDC for an additional 48 h (C and D). Cells were stained with DAPI and anti-phospho-H3 antibody, and photographs were taken (×600 magnification) (C). Cell proliferation was evaluated by percentage of cells with phospho-H3-positive nuclei. D, data are means ± S.D. of three independent experiments conducted in triplicate and expressed as the percentage of control or total cell number. ^*, p < 0.05.

FIGURE 2.

Effect of MDC on serum-induced TGase activity in cultured RPTC. RPTC were cultured in the medium containing 5% FBS in the absence or presence of MDC for 24 h at the indicated concentrations, and then 1 mm (5-(biotinamido)pentylamine) BP was added to the culture and incubated for an additional 1 h. Cells were harvested for analysis of TGase transamidation activity, as described under “Materials and Methods.” A representative blot from three independent experiments is shown (A). The biotin-labeled proteins were quantified by densitometry and normalized to actin B, data are means ± S.D. of three independent experiments and expressed as percentage of expression level relative to control. ^*, p < 0.05.

FIGURE 3.

Expression of TGases in RPTC. RPTC were cultured to 60-70% confluence. Then cell lysates were prepared and subjected to immunoblot analysis using antibodies to TGase-1, TGase-2, TGase-5, TGase-7, or actin (A). RPTC were fixed and then stained using antibodies against TGase-1 and E-cadherin followed by Alexa Fluor 488-conjugated anti-rabbit IgG, or Texas Red goat anti-rabbit IgG and DAPI (B). Representative photographs were taken from a nonconfluent (upper row) and a confluent area (lower row).

FIGURE 4.

Effect of siRNA specific to TGase-1 on RPTC proliferation. RPTC were transfected with scrambled siRNA or siRNA specific to TGase-1 and then cultured for 48 h in DMEM with 0.5% FBS. A, cells were stimulated with 5% FBS for 1 h in the presence of 1 mm BP. TG transamidation activity was analyzed as described in the legend to Fig. 2. B, cells were incubated for an additional 48 h in the presence of 5% FBS, and cell number was counted. C, cells were incubated for an additional 48 h in the presence of 5% FBS and cell proliferation was evaluated by counting cells with phospho-H3 staining. Data are means ± S.D. of three independent experiments conducted in triplicate and expressed as the percentage of cells treated with scrambled siRNA or total cell number. ^*, p < 0.05.

FIGURE 5.

Effect of MDC on phosphorylation of Akt, and Stat-3 in RPTC. A, RPTC were serum-starved for 24 h and then stimulated with 5% FBS for 1 h (A) or 4 and 8 h (B) in the presence or absence of 100 μm MDC. Cell lysates were analyzed by immunoblot analysis with antibodies against phospho-Akt, Akt, phospho-Stat-3, or Stat-3. C, RPTC were transfected with scrambled siRNA or siRNA specific to TGase-1 and then cultured for an additional 48 h in the presence of 0.5% FBS. After stimulation with 5% FBS for 1 h, cell lysates were analyzed by immunoblot analysis with antibodies against phospho-Akt, Akt, phospho-Stat-3, or Stat-3. Representative immunoblots from three or more experiments are shown.

FIGURE 6.

Effect of the JAK/Stat-3 pathway inhibition on RPTC proliferation. RPTC were cultured for 24 h in the presence of 5% FBS and then treated with AG490 at the indicated concentrations for an additional 24 h (A) or 1 h (B). A, cell proliferation was assessed by the MTT assay. Data are means ± S.D. ^*, p < 0.05, compared with control group. B, cell lysates were subjected to immunoblot analysis with antibodies against phospho-Stat-3 and Stat-3. Representative immunoblots from three or more experiments are shown. RPTC were transfected with scrambled siRNA or siRNA specific to JAK2 or Stat-3 and then cultured for 48 h in DMEM with 0.5% FBS (C-F). Cells were incubated for an additional 48 h in the presence of 5% FBS, and cell proliferation was evaluated by the MTT assay (C and E). Cells were stimulated with 5% FBS for 1 h, and cell lysates were analyzed by immunoblot analysis with antibodies against phospho-Stat-3, JAK2 (D), Stat-3, or actin (F).

FIGURE 7.

TGase-1 coexpression enhances JAK2-dependent phosphorylation of Stat-3. RPTC were transiently transfected with plasmids encoding wild types of TGase-1, JAK2, or Stat-3, either alone or in different combinations as indicated. After 48 h, the cells were harvested and Stat-3 was immunoprecipitated (IP) with an anti-Stat-3 antibody. Immunoprecipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. Representative immunoblots from three experiments are shown.

FIGURE 8.

Inhibition of TGase-1 disrupts the interaction of TGase-1 with JAK2 but does not affect serum-induced JAK2 phosphorylation. A and B, RPTC were transiently transfected with plasmids encoding TGase-1 and JAK2. TGase-1 was immunoprecipitated (IP) with an anti-TGase-1 antibody and then analyzed with antibodies against JAK2 or TGase-1 (A). JAK2 was immunoprecipitated with an anti-JAK2 antibody and then analyzed with antibodies against TGase-1 or JAK2 (B). RPTC were treated with MDC (100 μm) for 1 h (C) or TGase-1 siRNA for 48 h (D) and then harvested for immunoblot analysis with antibodies to phospho-JAK2 or JAK2. Representative immunoblots from three experiments are shown.

FIGURE 9.

The role of TGase-1 in serum-stimulated nuclear accumulation of Stat-3 and Stat-3 transcriptional activity. A, RPTC were cultured for 24 h in DMEM/F-12 with 5% FBS in the presence or absence of 100 μm MDC and then stained with anti-Stat-3 antibody and DAPI. B, RPTC were transfected with p-IRF1-SIE-Luc and serum-starved for 24 h and then incubated with DMEM alone, DMEM plus 5% FBS, or DMEM plus 5% FBS containing 100 μm MDC or 10 μm AG490 for an additional 24 h. C, RPTC were transfected with plasmids encoding wild types of TGase-1, JAK2, or Stat-3, either alone or in different combinations as indicated and then incubated for 24 h with DMEM plus 5% FBS. Cells were lysed, and equal amounts of protein were used to evaluate the luciferase activity by dual luciferase reporter assay as described under “Materials and Methods.” Data are means ± S.E. of three independent experiments conducted in triplicate and expressed as -fold increase relative to controls. ^*, p < 0.05.

FIGURE 10.

Effect of MDC, AG490, and Stat-3 siRNA on cyclin D1 and cyclin E expression. A and B, RPTC were serum-starved for 24 h and then incubated with 5% FBS for an additional 24 h in the presence or absence of 100 μm MDC (A) or 10 μm AG490 (B). C, RPTC were transfected with scrambled siRNA or siRNA specific to TGase-1 and then cultured for 48 h in DMEM with 5% FBS. Cell lysates were subjected to immunoblot analysis with antibodies against cyclin D1 and cyclin E. Representative immunoblots from three or more experiments are shown.