Figure 1.
Effects of HILI on TGF-β signaling at the level of Smad phosphorylation and ultimate effect on apoptosis.
A. HILI reduces expression of, PAI-1, and Smad7 and prevents Smad2/3 activation. HEK-293 cells were transfected with Myc-HILI at a concentration gradient (0.5 µg, 0.5 µg, 1.5 µg, and 3 µg per well) and treated with TGF-β for 1 h before cells were harvested as indicated. Cells lysates were used for Western blot analysis with anti-, anti-PAI-1, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2/3, anti-Smad4, anti-Smad7, anti-GAPDH, and anti-Myc antibodies. B. siHILI reverses the reduction of p21, PAI-1, Smad7, and phosphorylated Smad2/3 mediated by HILI. HEK-293 cells were transfected with siHILI or Myc-HILI, after 48 h, treated with TGF-β for 1 h before harvesting. They were then used in Western blot analysis by using antibodies as in (A). NC represents negative control transfected with siControl. C and D. HILI inhibits TGF-β-induced cell apoptosis. HEK-293 cells were respectively transfected with pcDNA3.1, Myc-HILI, siControl and siHILI, and treated with TGF-β for 12 h before cells were harvested as indicated. After transfection 48 h, apoptosis of cells was analyzed with Annexin V/PI double staining and flow cytometry. NC represents negative control without any treatments. Each experiment was performed in triplicate. *indicates P<0.05.
Figure 2.
Effects of HILI on degradation of TβRs and Smad. A.
HILI-mediated loss of TGF-β-induced Smad activation is restored by MG132. HEK-293 cells were transfected with Myc-HILI or pcDNA3.1. After 48 h, they were treated with MG132 for 6 h. TGF-β was added for the last 1 h, as indicated. Cells were harvested for Western blotting using anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2/3, anti-Smad4, anti-GAPDH, and anti-Myc antibodies. B. HILI promotes TβR degradation. HEK-293 cells were transfected with Myc-HILI. After 48 h, the cells were treated as in (A). Cell lysates were used for Western blotting with anti-TβRII, anti-TβRI, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2/3, anti-Smad4, anti-Hsp90, anti-GAPDH, and anti-Myc antibodies. C. siHILI improves TβR protein levels. HEK-293 cells were transfected with siControl or siHILI. After 48 h, cell lysates were used for Western blotting with anti-TβRII, anti-TβRI, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2/3, anti-GAPDH, and anti-HILI antibodies. D and E. Neither HILI nor siHILI was found to affect TβR mRNA levels. HEK-293 cells were transfected with pcDNA3.1, Myc-HILI, siControl, and siHILI as indicated. After 48 h, the cells were harvested for total RNA extraction and then for quantitative RT-PCR. Each experiment was performed in triplicate. N.S. indicates P>0.05.
Figure 3.
Effects of interaction between HILI and Hsp90 on the formation of Hsp90-TβR complexes.
A and B. HILI impairs interaction between Hsp90 and TβRs. HEK-293 cells were transfected with Myc-HILI at a concentration gradient (1.5 µg and 3 µg per well). After 48 h of culture, cells were treated with MG132 for 6 h where indicated. The lysates were subjected to anti-Hsp90 or anti-TβRs (TβRII, TβRI) IP. Interactions between Hsp90 and TβRs were detected by (A) anti-Hsp90 and anti-TβRII or (B) anti-TβRI immunoblotting. Myc-HILI expression was detected using anti-Myc. TCL indicates total-cell lysates. C–I. HILI interacts with Hsp90 in vivo. C. Exogenous Myc-HILI binds HA-Hsp90. HEK-293 cells were cotransfected with Myc-HILI and HA-Hsp90. After 48 h of cell culture, Myc-HILI and HA-Hsp90 were coimmunoprecipitated (Co-IP) by anti-Myc or anti-HA. Then Western blotting was using to evaluate any interactions. D. Both HILI and Hsp90 are localized in cytoplasm. HEK-293 cells were transfected as in (C). The cells were harvested for immunofluorescence with anti-Myc and anti-HA antibodies. Scale bars, 10 µm. E. Endogenous interactions between HILI and Hsp90 in HEK-293 cells. Co-IP was performed with anti-HILI or anti-Hsp90, followed by Western blotting. F. The bands of Hsp90 and HILI are specific. HEK-293 cells were transfected with siControl or siHILI. The cell lysates were used for Co-IP by anti-HILI and anti-Hsp90. G. Different HILI deletion mutants. We constructed various HILI mutants by segmented-PCR and fusion PCR. All mutants were cloned into expression vector pcDNA3.1+Myc. H and I. Interaction between different HILI mutants and Hsp90. HEK-293 cells were cotransfected with Myc-HILI deletion mutants and HA-Hsp90. Co-IP as in (C). J. HILI interacts directly with Hsp90 in vitro. In vitro cell-free HILI and Hsp90 protein expression were carried out in two reactions using TnT® system, separately. The HILI and Hsp90 TnT® reactions were mixed together in binding buffer with protease inhibitors and incubated on a rotating platform at 4°C for 3h. Interaction between single HILI and Hsp90 protein was detected by Co-IP and Western blotting using anti-HILI and anti-Hsp90. K. Reduction of TβRs mediated by HILI was rescued by overexpression of Hsp90. Cell lysates were used for Western blotting with anti-TβRII, anti-TβRI, anti-GAPDH, anti-Myc, and anti-HA. L. siHILI increases TβR levels, but siHsp90 reduces them. HEK-293 cells were transfected with siControl, siHILI, and siHsp90 as indicated. After 48 h, cell lysates were used for Western blotting with anti-TβRII, anti-TβRI, anti-GAPDH, anti-HILI, and anti-Hsp90.
Figure 4.
Role of smurf2 is required for HILI-regulated TβR degradation.
A. HILI improves TβRI ubiquitination. HEK-293 cells were cotransfected with Myc-HILI and HA-Ubiquitin, after 48 h, treated with MG132 for 6 h, TGF-β for the last 1 h as indicated. TβRI was precipitated with anti-TβRI antibody, then ubiquitination and degradation of TβRI were determined by anti-HA (Ub) and anti-TβRI immunoblotting. B. Smurf2 knockdown abolishes HILI-regulated TβRI ubiquitination. HEK-293 cells were cotransfected with Myc-HILI, HA-Ubiquitin, siSmurf2, and siControl where indicated. TβRI ubiquitination was checked as in (A). C. Smurf2 knockdown blocks HILI-regulated TβR degradation. HEK-293 cells were cotransfected with Myc-HILI, siSmurf2 and siControl where indicated. After 48 h, the lysates were used for Western blotting by anti-TRβII, anti-TβRI, anti-GAPDH, anti-Smurf2, and anti-Myc.
Figure 5.
Inhibitive effects of HILI on the TGF-β signaling.
HILI competes with TβRs for Hsp90 by binding molecular chaperones, preventing the formation of Hsp90-TβR complexes, and promoting the ubiquitination and degradation of TβRs dependent on the ubiquitin E3 ligase Smurf2. This terminates TGF-β signaling.