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. 2017 Sep 19;114(38):10113-10118.
doi: 10.1073/pnas.1705755114. Epub 2017 Sep 5.

Smad7 enables STAT3 activation and promotes pluripotency independent of TGF-β signaling

Yi Yu  1   2 , Shuchen Gu  1 , Wenjian Li  1 , Chuang Sun  2   3   4 , Fenfang Chen  1 , Mu Xiao  1 , Lei Wang  1 , Dewei Xu  1 , Ye Li  1 , Chen Ding  5   6 , Zongping Xia  1 , Yi Li  3   7 , Sheng Ye  1 , Pinglong Xu  1 , Bin Zhao  1 , Jun Qin  5   8 , Ye-Guang Chen  9 , Xia Lin  2   3   4 , Xin-Hua Feng  10   2   3   4
Affiliations

Smad7 enables STAT3 activation and promotes pluripotency independent of TGF-β signaling

Yi Yu et al. Proc Natl Acad Sci U S A. .

Abstract

Smad7 is a negative feedback product of TGF-β superfamily signaling and fine tunes a plethora of pleiotropic responses induced by TGF-β ligands. However, its noncanonical functions independent of TGF-β signaling remain to be elucidated. Here, we show that Smad7 activates signal transducers and activators of transcription 3 (STAT3) signaling in maintaining mouse embryonic stem cell pluripotency in a manner independent of the TGF-β receptors, yet dependent on the leukemia inhibitory factor (LIF) coreceptor glycoprotein 130 (gp130). Smad7 directly binds to the intracellular domain of gp130 and disrupts the SHP2-gp130 or SOCS3-gp130 complex, thereby amplifying STAT3 activation. Consequently, Smad7 facilitates LIF-mediated self-renewal of mouse ESCs and is also critical for induced pluripotent stem cell reprogramming. This finding illustrates an uncovered role of the Smad7-STAT3 interplay in maintaining cell pluripotency and also implicates a mechanism involving Smad7 underlying cytokine-dependent regulation of cancer and inflammation.

Keywords: STAT3; Smad7; TGF-β; differentiation; gp130; pluripotency.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Smad7 promotes self-renewal and inhibits differentiation of ESCs. (A) Smad7 is down-regulated during EB formation in CGR8 cells. qRT-PCR was used to analyze mRNA levels of Smad7, Oct4, Sox2, Nanog, and GAPDH. Data are shown as mean ± SEM; n = 3. (B) Smad7 maintains a high expression level of pluripotency markers during EB formation. SFB–Smad7–tet-on CGR8 underwent EB differentiation for 4 d in the absence or presence of 1 μg/mL Dox. Total RNAs were subjected to qRT-PCR to examine expression levels of Oct4, Nanog, Sox2, and Smad7. Data are shown as mean ± SEM; n = 3. *P < 0.05. (C) Smad7 inhibits ESC differentiation during EB formation. The experiment was essentially performed as described in Fig. 1B, and qRT-PCR was used to examine mRNA levels of ectoderm, mesoderm, and endoderm markers. Data are shown as mean ± SEM; n = 3. *P < 0.05.
Fig. S1.
Fig. S1.
Smad7 promotes self-renewal and inhibits differentiation of ESCs. (A) Smad7 is down-regulated during EB formation in CGR8 cells. Cell lysates were subjected to Western blot analysis to examine protein levels of Smad7, Oct4, Sox2, Nanog, and GAPDH (as a negative control) with appropriate antibodies. (B) Differentiation markers are up-regulated during EB formation in CGR8 cells. Total RNAs were subjected to qRT-PCR to examine mRNA levels of Cxcl12, Brachyury/T, and Foxa2. Data are shown as mean ± SEM; n = 3. (C) Dox induces an approximately two-fold increase in Smad7 mRNA in SFB–Smad7–tet-on CGR8 cells. Wild-type and SFB–Smad7–tet-on CGR8 cells were cultured in the absence or presence of 1 μg/mL Dox for 72 h. Total RNAs were extracted from cells and subjected to qRT-PCR to examine expression levels of Smad7. Data are shown as mean ± SEM; n = 3. *P < 0.05. (D) Dox induction of Smad7 gives a moderate increase in total Smad7 protein in SFB–Smad7–tet-on CGR8 cells. Wild-type and SFB–Smad7–tet-on CGR8 cells were cultured in the absence or presence of 1 μg/mL Dox for 72 h. Cells were subjected to Western blot analysis to examine the protein level of Smad7. (E) Dox treatment maintains the mRNA level of Smad7 during EB formation. SFB–Smad7–tet-on CGR8 underwent EB differentiation for 4 d in the absence or presence of 1 μg/mL Dox. Total RNAs were extracted from cells and subjected to qRT-PCR analysis to examine the mRNA level of Smad7. Data are shown as mean ± SEM; n = 3. *P < 0.05.
Fig. 2.
Fig. 2.
Smad7 is essential in maintenance of pluripotency. (A–C) Depletion of Smad7 down-regulates expression of pluripotency markers and induces differentiation in ESCs. CGR8 cells stably expressing shSmad7 were established as described in Supporting Information. Smad7r is a RNAi-resistant variant of Smad7. (A) qRT-PCR analysis of Oct4, Nanog, Sox2, and Smad7. Data are shown as mean ± SEM; n = 3. (B) Immunofluorescence analysis of Oct4. DNA was stained with DAPI. (C) AP staining of CGR8 cells stably expressing shSmad7 and/or Smad7r. (D) Depletion of Smad7 enhances ESC differentiation into ectoderm and mesoderm, but not endoderm or trophectoderm. qRT-PCR was used to examine mRNA levels of indicated differentiation markers. Data are shown as mean ± SEM; n = 3. *P < 0.05, **P < 0.01. (E and F) Smad7 depletion inhibits the reprogramming efficiency in reprogrammable MEFs. Reprogrammed iPSC colonies were identified by AP staining (E) and quantitation of GFP-positive clones (F) at day 14. Data are shown as mean ± SEM; n = 3. **P < 0.01.
Fig. S2.
Fig. S2.
Smad7 is essential in maintenance of pluripotency. (A) Stable knockdown of Smad7 in CGR8 cells. Cell lysates from pLKO.1–control and pLKO.1–shSmad7 CGR8 stable cells were subjected to qRT-PCR and Western blot analysis. qRT-PCR data are shown as mean ± SEM; n = 3. **P < 0.01. (B) shSmad7 down-regulates expression of pluripotency marker proteins in ESCs. pLKO.1–control and pLKO.1–shSmad7 CGR8 stable cells were transfected with the indicated plasmids for 48 h. Cells were subjected to Western blot analysis to examine protein levels of Nanog, Sox2, Oct4, Smad7, and β-actin. (C) Schematic representation of the FLAG-tagged Smad7r expression vector. The shSmad7 _target sequence (encoding amino acids 893–913, above the nucleotide sequence) is indicated by capital letters. Mutations introduced are indicated by bold/italic letters. (D) pLKO.1–control and pLKO.1–shSmad7 CGR8 stable cells were transfected with the empty vector or the indicated Smad7 expression vectors. Cell lysates were subjected to Western blot analysis to examine Smad7 protein levels. Smad7-WT is the FLAG-tagged wild-type Smad7, whereas Smad7r is a FLAG-tagged Smad7 variant resistant to shSmad7. (E) Depletion of Smad7 enhances expression of Cxcl12 (ectodermal marker) and Brachyury/T (mesodermal marker), but not Foxa2 (endodermal marker). Cell lysates of shSmad7 and control ESCs were subjected to Western blot analysis to examine protein levels of Cxcl12, T, and Foxa2. (F) Knockdown of Smad7 inhibits stemness and enhances ES cell differentiation into ectoderm and mesoderm. CGR8 cells were transfected with different concentrations of Smad7 siRNA as indicated and cultured for 48 h. Cell lysates were subjected to Western blot analysis to examine protein levels of pluripotency and differentiation markers. (G) Smad7 increases during the MEF reprogramming process. Cells were collected every 3 d from day 0 to day 15 after infection with pMXs-based OSKM. Total RNAs were extracted from cells and subjected to qRT-PCR to examine expression levels of Smad7, Oct4, and Sox2. Data are shown as mean ± SEM; n = 3. *P < 0.05.
Fig. 3.
Fig. 3.
Smad7 promotes pluripotency independent of canonical TGF-β/Smad signaling. (A) Smad7 promotes ESC self-renewal independent of TGF-β/BMP signaling. SFB–Smad7–tet-on cells were pretreated with 5 μM SB431542 (TGFBRi) or 10 μM Dorsomorphin (BMPRi) for 12 h and then cultured in indicated medium for another 3 d. qRT-PCR was used to analyze expression of indicated pluripotency markers. Data are shown as mean ± SEM; n = 3. (B) Inhibition of TGF-β/BMP signaling does not reverse the effect of siSmad7 in ESC pluripotency. CGR8 cells were transfected with 40 pM Smad7 siRNA or control siRNA and cultured with TGFBRi or BMPRi for 2 d. Cell lysates were subjected to Western blot analysis.
Fig. S3.
Fig. S3.
Smad7 promotes pluripotency independent of canonical TGF-β/Smad signaling. (A) siSmad7 inhibits Oct4 expression independent of TGF-β/BMP signaling. CGR8 cells were transfected with 40 pM Smad7 siRNA or control siRNA and then split into six-well plates at a low density. Transfected cells were treated with TGFBRi or BMPRi for 3 d. Cells were fixed and immune stained with anti-Oct4 antibody. DNA was stained with DAPI. (B) siSmad7 inhibits ES cell colony formation independent of TGF-β/BMP signaling. CGR8 cells were transfected with siRNAs as described in Fig. S3A. Transfected cells were treated with TGFBRi or BMPRi for 5 d, and then fixed and subjected to AP staining. (C) Smad7 mutant K401E does not inhibit the TGF-β–induced SBE–luc reporter activity. HaCaT cells were cotransfected with wild-type Smad7 (WT) or Smad7–K401E together with the Smad-binding element (SBE)–luc reporter (a synthetic TGF-β–responsive reporter), followed by stimulation with 5 μM SB431542 (TGF-β type I receptor inhibitor) or 2 ng/mL TGF-β for 12 h. Cells were harvested for luciferase assay. Data are shown as mean ± SEM; n = 3. *P < 0.05. (D) Smad7 mutant K401E does not inhibit the BMP-induced Id–luc reporter activity. C2C12 cells were cotransfected with wild-type Smad7 (WT) or Smad7–K401E together with the expression plasmid for Id1–luc reporter (a luciferase reporter driven by the Id1 promoter containing the BMP-responsive element), followed by stimulation with 10 μM Dorsomorphin (BMP type I receptor inhibitor) or 50 ng/mL BMP2 for 12 h. Cells were harvested for luciferase assay. Data are shown as mean ± SEM; n = 3. *P < 0.05. (E) Smad7 promotes ESC stemness independent of its ability to bind to TβRI or BMPRI in ESCs. CGR8 cells were transfected with FLAG–Smad7 (WT), the K401E mutant or control plasmid and then treated with or without 0.1 ng/mL LIF for 48 h. Total RNAs were extracted from cells and subjected to qRT-PCR to examine expression level of indicated pluripotency markers. Data are shown as mean ± SEM; n = 3. *P < 0.05.
Fig. 4.
Fig. 4.
Smad7 potently activates gp130-mediated STAT3 signaling. (A) Smad7 stimulates LIF-induced STAT3 activation in ESCs. CGR8 control (Ctrl) and SFB–Smad7–tet-on cells were cultured ± Dox (1 μg/mL, 72 h) and then treated with LIF (0.1 ng/mL, 20 min). Cell lysates were subjected to Western blot analysis. (B) Smad7 enhances SOCS3 expression in ESCs. SFB–Smad7–tet-on or control cells were treated with or without 1 μg/mL Dox for 72 h. qRT-PCR was used to examine the SOCS3 mRNA level. Data are shown as mean ± SEM; n = 3. *P < 0.05. (C) Smad7 activates LIF-induced STAT3 signaling independent of TβRI or BMPRI in ESCs. WT and the K401E mutant of Smad7 are indicated. CGR8 cell transfection, LIF treatment, and Western blot analysis were done as described in Supporting Information. (D) Inhibition of TβRI/BMPRI does not influence the effect of siSmad7 in LIF-induced STAT3 signaling in ESCs. CGR8 cell transfection, LIF treatment, and Western blot analysis were done as described in Supporting Information.
Fig. S4.
Fig. S4.
Smad7 potentiates gp130-mediated STAT3 signaling. (A) Smad7 enhances LIF-induced STAT3 activation and Oct4 expression in ES cells. SFB–Smad7–tet-on cells were cultured with or without 1 μg/mL Dox for 72 h and then treated with 0.1 ng/mL LIF for indicated time. Cell lysates were subjected to Western blot analysis. (B) Smad7 increases the sensitivity of ES cells to LIF-induced STAT3 activation. SFB–Smad7–tet-on cells were cultured with or without 1 μg/mL Dox for 72 h and then treated with indicated concentrations of LIF for 20 min. Cell lysates were subjected to Western blot analysis. (C) Smad7 up-regulates LIF-activated reporter activity in ESCs. SFB–Smad7–tet-on or control cells were transfected with M67–luc reporter (a synthetic luciferase reporter with STAT3-responsive elements) and Renilla luciferase plasmids, and then treated with or without 1 μg/mL Dox for 72 h. Cell were harvested for luciferase assay. Data are shown as mean ± SEM; n = 3. *P < 0.05. (D) Inhibition of TβRI/BMPRI does not affect the ability of Smad7 in enhancing LIF-induced STAT3 signaling in ESCs. SFB–Smad7–tet-on cells were pretreated with TGFBRi or BMPRi for 12 h and then cultured with or without 1 μg/mL Dox for 72 h and then treated with 0.1 ng/mL LIF for another 20 min. Cell lysates were subjected to Western blot analysis.
Fig. 5.
Fig. 5.
Smad7 directly competes with SHP2/SOCS3 for gp130 binding and enables STAT3 signaling in maintaining pluripotency. (A) Smad7 interacts with gp130 under physiological conditions. Immunoprecipitation and Western blot analysis were done as described in Supporting Information. (B) Smad7 directly interacts with gp130. In vitro binding was carried out with purified His–gp130–ICD and in vitro translated Smad7. Experimental details are described in Supporting Information. (C) Smad7 displaces SHP2 on gp130. Increasing concentrations of purified His–Smad7 proteins were added to the gp130Y759E–SHP2 complex, and followed by Western blot analysis with indicated antibodies. (D) Smad7 competes with endogenous SHP2 and SOCS3 for gp130 binding in CGR8 cells. Cell lysates were immunoprecipitated with anti-SOCS3 antibody or anti-SHP2 antibody. Cell culture, LIF treatment, immunoprecipitation, and Western blot analysis were done as indicated and described in Supporting Information. (Left) SFB–Smad7–tet-on cells treated with or without Dox; (Right) CGR8 cells with shCtrl and shSmad7. In the bottom blots, FLAG/Smad7 means the use of anti-FLAG in lanes 1–4 and anti-Smad7 in lanes 5–8. (E) Smad7 overcomes SHP2- or SOCS3-mediated suppression of ESC colony formation. CGR8 cell transfection and AP staining were performed as described in Supporting Information. The bar graph represents the fold change of numbers of uniform AP+ colonies in Fig. S6F. Data are shown as mean ± SEM; n = 3. *P < 0.05. (F) SiSmad7 inhibition of ESC self-renewal is reversed by simultaneous knockdown of SHP2 and SOCS3. Experiments and data analysis were done as described in Fig. 5E and Supporting Information. The bar graph represents the fold change of numbers of uniform AP+ colonies in Fig. S6H. Data are shown as mean ± SEM; n = 3. *P < 0.05. (G) A working model for Smad7 potentiating STAT3 activation. (Left) LIF and related cytokines (C) bind to the gp130 receptor complex. Receptor-associated JAK kinases phosphorylate STAT3 leading to STAT3 accumulation in the nucleus, where STAT3 controls expression of _target genes, including Smad7 and SOCS3. SOCS3 and SHP2 bind to gp130 to inhibit STAT3 activation. Smad7 can compete for the gp130 binding, maintaining STAT3 activation. (Right) Active and inactive forms of the cytokine-receptor–gp130 complex are shown.
Fig. S5.
Fig. S5.
Smad7 directly binds to the cytoplasmic domain of gp130 and disrupts the binding of SHP2/SOCS3 to gp130. (A) Smad7 interacts with gp130 in vivo. HEK293T cells were cotransfected with FLAG–Smad7 and HA–gp130 and then treated with 1 ng/mL LIF for 2 h before harvest. The cell lysates were harvested and immunoprecipitated with anti-HA antibody. Smad7 and gp130 were detected from the immunoprecipitates by Western blot analysis. (B) The MH2 domain (or C domain) of Smad7 binds to gp130. HEK293T cells were transfected with indicated plasmids. Levels of these proteins in IP products and whole cell lysates were analyzed by Western blot analysis. (C) The 734–764 aa region in the cytoplasmic domain of gp130 is essential in Smad7 binding. HEK293T cells were cotransfected with FLAG–Smad7 and a HA-tagged gp130 wild-type or a deletion mutant, and then treated with 1 ng/mL LIF for 2 h before harvest. Cell lysates were harvested and immunoprecipitated with anti-FLAG antibody. Smad7 and gp130 variants were detected from the immunoprecipitates by Western blot analysis. (D) Formation of the Y759E–SHP2 complex in E. coli. Fusion proteins of GST–gp130–Y759E and His–SHP2 were coexpressed in E. coli. The Y759E–SHP2 complex was retrieved by glutathione Sepharose beads, as indicated by Coomassie staining. P, purified proteins on glutathione Sepharose beads; W, whole cellular protein lysates.
Fig. S6.
Fig. S6.
Smad7 stimulates stemness through STAT3 activation. (A) STAT3 depletion blocks the positive effect of Smad7 in ESC colony formation. SFB–Smad7–tet-on cells were transfected with 40 pM STAT3 siRNA or control siRNA and then split into six-well plates at a low density. Transfected cells were cultured with or without 1 μg/mL Dox for 5 d and then fixed and subjected to AP staining. (B) STAT3 depletion blocks the effect of Smad7 in ESC pluripotency. SFB–Smad7–tet-on cells were transfected with 40 pM STAT3 siRNA or control siRNA and then cultured with or without 1μg/mL Dox for 3 d. Cell lysates were subjected to Western blot analysis. (C) siSmad7 inhibition of ESC self-renewal is rescued by constitutive activation of STAT3 signaling. CGR8 cells were cotransfected with Smad7 siRNA (40 pM) and FLAG–STAT3C and then split into six-well plates at a low density. Transfected cells were cultured with or without 1 ng/mL LIF for 5 d and then fixed and subjected to AP staining. The bar graph displays the fold change of numbers of colonies with uniform AP staining. Data are shown as mean ± SEM; n = 3. *P < 0.05. (D) Constitutive active STAT3C reverses the effect of siSmad7 inhibition on pluripotency marker expression. CGR8 cells were cotransfected with Smad7 siRNA (40 pM) and FLAG–STAT3C and then cultured with or without 1 ng/mL LIF for 2 d. Total RNAs were extracted from cells and subjected to qRT-PCR to examine mRNA levels of indicated pluripotency markers. (E) Inhibition of JAK kinase activity attenuates the effect of Smad7 in ES cell pluripotency. SFB–Smad7–tet-on cells were treated with 20 nM JAK inhibitor Filgotinib for 12 h and then cultured with or without 1 μg/mL Dox and 0.1 ng/mL LIF for 72 h. Cell lysates were subjected to Western blot analysis. (F) Smad7 overcomes the SHP2/SOCS3-mediated suppression of ESC colony formation. CGR8 cells were transfected with FLAG–Smad7, FLAG–SHP2, and/or MYC–SOCS3 as indicated. Experiments were performed as described in Fig. S6A. Arrowheads point to ESC colonies that are not uniformly ALP+. Statistical analysis of numbers of colonies with uniform ALP staining is presented in Fig. 5E. (G) Smad7 antagonizes the suppressive effects of SHP2 and SOCS3 on STAT3 activation and ESC pluripotency. CGR8 cells were transfected with FLAG–Smad7, FLAG–SHP2, and/or MYC–SOCS3 as indicated. Cell lysates were subjected to Western blot analysis. (H) siSmad7 inhibition of ESC self-renewal is reversed by simultaneous knockdown of SHP2 and SOCS3. CGR8 cells were transfected with siRNA (40 pM) specific to Smad7, SHP2, and/or SOCS3 as indicated. Experiments were performed as described in Fig. S6A. Statistical analysis of numbers of colonies with uniform ALP staining is presented in Fig. 5F. (I) siSmad7 inhibition of ES cell self-renewal is rescued by simultaneous knockdown of SHP2 and SOCS3. CGR8 cells were transfected with siRNA (40 pM) specific to Smad7, SHP2, or SOCS3 as indicated for 2 d. Cell lysates were subjected to Western blot analysis.
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