Article Figures & Data
Figures
- FIG. 1.
Expression analysis of mPHIP in pancreatic β cells and insulin-producing β-cell lines. (A) Schematic representation of PHIP1 translation products illustrates the presence of eight WD40 motifs spanning residues 172 to 498 (black boxes), two bromodomains (BD) located between residues 1158 and 1423 (gray boxes), and two NLS at positions 912 to 924 and 1438 to 1465 (thick bar). Alternative splicing of mPHIP predicts multiple transcript variants that can be recognized by raised anti-PHIP antibodies. mPHIP variant 2 (GenBank accession number XP_900529) and variant 11 (GenBank accession number XP_900632) produce proteins that retain six and one of the eight WD40 repeats, respectively, whereas mouse variant 9 (GenBank accession number XP_900614.1), which we previously cloned, is lacking WD40 repeats. (B) Pancreatic β cells express a prominent 206-kDa form of PHIP. Total proteins (50 μg) from freshly extracted mouse islets and insulin-producing cell lines INS-1 and MIN6 were fractionated on a 6% sodium dodecyl sulfate-polyacrylamide gel and immunoblotted with anti-PHIP antibodies. (C) qPCR analysis of PHIP1 mRNA expression in various mouse tissues and MIN6 cells was performed with primers specific for mPHIP1. Results are expressed as the means ± standard errors of the means for three independent experiments done in triplicate. WAT, white adipose tissue; BAT, brown adipose tissue. (D) Anti-PHIP antibodies specifically detect two recombinant isoforms of PHIP. MIN6 cells were transiently transfected with either pcDNA3 control or the pcDNA3-HA-hPHIP1 or pcDNA3-HA-mPHIP vectors encoding the 1,821-aa and 902-aa PHIP isoforms, respectively. Cellular lysates were collected 48 h posttransfection and immunoblotted with anti-PHIP antibodies. Exogenously expressed HA-hPHIP1 and HA-mPHIP are denoted by arrowheads. Endogenous mPHIP1 is shown by an arrow (→). (E) siRNA knockdown of PHIP in INS-1 cells. INS-1 cells were transfected with 50 nM of siRNA pool to rat PHIP1 or nonsilencing scrambled (Scr) siRNAs. Immunoblot analysis shows knockdown of 206- and ∼145-kDa immunoreactive signals representing possible transcript variants of PHIP1. Actin was used as a loading control. IB, immunoblot.
- FIG. 2.
Alignment of deduced aa sequences of cloned hPHIP1 (GenBank accession number DQ924532) and predicted mPHIP1 (GenBank accession number XP_358384) isoforms reveals 96% aa identity (highlighted in black).
- FIG. 3.
PHIP is localized to the nucleus of pancreatic β cells. (A) Section of mouse pancreas stained with anti-PHIP (red) and 4′,6′-diamidino-2-phenylindole (DAPI; blue) demonstrates predominant PHIP expression in nuclei of islet cells. Original magnification, ×100. (B) Five-micrometer consecutive sections of mouse pancreas were stained with anti-PHIP (green), anti-insulin (red), antiglucagon (green), and anti-PDX-1 (red) antibodies as described in Materials and Methods and counterstained with hematoxylin and eosin (H&E). The specificity of staining was verified by the inclusion of appropriate preimmune antibodies as a negative control. Original magnification, ×200; scale bar, 20 μm. (C) Coimmunostaining of PHIP1 and glucagon in dispersed islets from MIP-GFP transgenic mice shows comparable levels of PHIP (blue) in β cells (green) and in glucagon-positive α cells (red). (D) Immunostaining of MIN6 cells with anti-PHIP (upper left panel, red) and anti-IRS2 (upper right panel, green) antibodies reveals differential subcellular localization for PHIP and IRS2. In the upper middle panel, MIN6 cells were infected with AdPHIP1 (MOI, 400) and localization of ectopically expressed PHIP1 (red) was detected by anti-HA antibodies 48 h postinfection. Note the exclusively nuclear anti-HA staining. DAPI counterstaining indicates localization of nuclei. Original magnification, ×400; scale bar, 20 μm. (E) Subcellular fractionation shows that endogenously and exogenously expressed PHIP1 localized in the nuclear fraction. INS-1 cells were infected at an MOI of 400 with AdPHIP1 and, 48 h afterwards, were collected, and 50 μg of WCL and cytosolic and nuclear fractions were loaded for Western blotting analysis. Levels of IRS2 and cyclin D2 were measured as a control for subcellular fractionation efficacy. (F) IGF-1 does not induce nuclear-cytoplasmic shuttling of PHIP1. After serum starvation (DMEM and 0.5% BSA) for 24 h, INS-1 and MIN6 cells were exposed for 16 h to 10 nM of IGF-1 in DMEM and 0.5% BSA (in the presence of IGF-1 [+IGF-1]) and immunostained for PHIP1 (red). Cells from control groups were exposed to medium containing 0.5% BSA only (in the absence of IGF-1 [−IGF-1]). DAPI counterstaining indicates localization of nuclei. Original magnification, ×400.
- FIG. 4.
Ad-mediated overexpression of PHIP1 promotes proliferation and potentiates IGF-1-stimulated mitogenesis of β cells. (A) Immunoblot analysis of PHIP1 expression in INS-1 cells infected with increasing doses of AdGFP and AdPHIP1 (MOI, 100 to 400, as indicated) indicates dose-dependent increase of exogenous PHIP1 expression (left). The IRS2 immunoblot demonstrates the level of IRS2 overexpression in AdIRS2-infected cells (right). Actin expression was used as a loading control. (B) Time course of INS-1 and NIH 3T3 proliferation assessed by MTS assay and counting of live cells, respectively, 24 to 96 h postinfection with AdGFP, AdPHIP1, and AdIRS2 (MOI, 200). Cells were incubated in the presence of 11 mM glucose-10% FBS. Experiments were performed three times in sextuplicate. Results are shown as means ± SD. *, P < 0.05 versus results for AdGFP-infected cells. OD450, optical density at 450 nm. (C) Ad-mediated overexpression of PHIP1 enhances IGF-1-dependent mitogenesis and promotes increase of cyclin D2 protein levels in INS-1 cells. Cells were infected with AdGFP, AdPHIP1, and AdIRS2 (MOI, 200) and, 16 h postinfection, were made quiescent for 24 h. Subsequently, cells were incubated in RPMI medium with 15 mM glucose in the presence (+) or absence (−) of 10 nM IGF-1. BrdU incorporation was measured as indicated in Materials and Methods. The data are expressed as the increases ± standard errors of the means compared to results for control cells (AdGFP-infected cells treated with 15 mM glucose in the absence of IGF-1 [GFP/−IGF group]). Experiments were performed three times in quadruplicate. *, P < 0.05. Bottom panel, cells were treated as described above and lysates were immunoblotted with cyclin D2 antibodies. Actin was used as a loading control. (D) Densitometry analysis from the three independent experiments described for panel C is summarized as a histogram. The data are expressed as the increases ± standard errors of the means. *, P < 0.05 versus results for AdGFP-infected cells treated in the absence of IGF-1; #, P < 0.05 versus results for AdGFP-infected cells treated in the presence of IGF-1. (E) Ad-mediated overexpression of PHIP1 enhances IGF-1-dependent cyclin D2 promoter activity in INS-1 cells. Cyclin D2 luciferase reporter was transfected into INS-1 cells. Subsequently, cells were infected with increasing MOIs, as indicated, of AdGFP or AdPHIP1 and incubated in the presence (+) or absence (−) of IGF-1 (10 nM). Luciferase assay was performed as indicated in Materials and Methods. Experiments were performed three times in triplicate. The data are expressed as the increases ± SD compared to results for control cells (AdGFP-infected cells treated with 15 mM glucose in the absence of IGF-1 [AdGFP/−IGF group]). *, P < 0.05 versus results for AdGFP group infected with same MOI in the absence of IGF-1; #, P < 0.05 versus results for AdPHIP1 group infected with same MOI in the presence of 10 nM of IGF-1. +, present; −, absent.
- FIG. 5.
Inhibition of PHIP1 expression blocks IGF-1- and IRS2-induced mitogenesis. INS-1 cells were transiently transfected with nonsilencing scrambled (SCR) and anti-PHIP1 (SiPHIP1) siRNAs and infected with AdGFP or AdIRS2 at an MOI of 200. (A) [3H]thymidine incorporation was measured 24 h after IGF-1 stimulation as indicated in Materials and Methods. Experiments were performed three times in triplicate. Results are shown as means ± SD. *, P < 0.05 versus results for cells transfected with SCR siRNA, infected with AdGFP (SCR + AdGFP), and stimulated with IGF-1 (+IGF); #, P < 0.05 versus results for cells transfected with SCR, transfected with AdIRS2 (SCR + AdIRS2), and not stimulated with IGF-1 (−IGF); **, P < 0.05 versus results for cells transfected with SCR siRNA, infected with AdIRS2, and stimulated with IGF-1. (B) Immunoblot analyses for PHIP1, IRS2, phospho-Ser473 PKB, cyclin D2, and actin in cells treated as described for panel A. The immunoblots are representative of at least two independent experiments. +, present; −, absent.
- FIG. 6.
Ad-mediated overexpression of PHIP1 prevents FFA-induced INS-1 cell apoptosis and induces PKB activation. (A) INS-1 cells were infected in six-well plates with AdGFP, AdPHIP1, and AdIRS2 (MOI, 75 and 400, as indicated). Sixteen hours postinfection, cells were incubated for 15 h with 15 mM glucose in the presence of 0.5% BSA alone or 0.4 mM of OA-0.5% BSA (OA). The incidence of apoptosis was assayed as described in Materials and Methods. Results are shown as the means ± SD for three independent experiments. *, P < 0.05 comparing results with AdPHIP1 to results with AdGFP in the absence of IGF-1 (−IGF-1); #, P < 0.05 comparing results with AdPHIP1 to results with AdGFP in the presence of IGF-1 (+IGF-1). (B) Ectopic PHIP1 overexpression induces PKB phosphorylation. WCL of AdGFP- and AdPHIP1-infected cells (MOI, 400) treated for 15 h with 15 mM glucose in the presence of 0.5% BSA and 10 ng/ml of IGF-1 were subjected to Western blot analysis for phospho-Ser473 (pSer473) and total PKB. Densitometry analysis from three independent experiments is summarized as a histogram. The data are expressed as n-fold increases ± standard errors of the means. *, P < 0.05 comparing results with AdPHIP1 to results with AdGFP in the absence of IGF-1; #, P < 0.05 comparing results with AdPHIP1 to results with AdGFP in the presence of IGF-1. +, present; −, absent. (C) Ectopic PHIP1 overexpression induces PKB phosphorylation and inhibits caspase-9 and -3 activation. WCL of AdGFP-, AdPHIP1-, and AdIRS2-infected (MOI, 400) INS-1 cells treated as described above were subjected to Western blot analysis for activated PKB (phospho-Ser473 [pSer473 PKB] and phospho-Thr308 [pThr308 PKB] PKB), total PKB, the proteolytically cleaved, activated forms of caspase-9 (Casp9) and -3 (Casp3), and Bax. Actin was used as a loading control. Representative immunoblots are shown. +, present; −, absent. (D) Densitometry analyses from the three independent experiments described for Fig. 6B are summarized as histograms. The data are expressed as n-fold increases ± standard errors of the means. Phospho-PKB levels were normalized to total PKB level, and cleaved procaspase-9 and -3 and Bax levels to actin level. *, P < 0.05 comparing results with AdPHIP1 to results with AdGFP in the absence of IGF-1; #, P < 0.05 comparing results with AdPHIP1 to results with AdGFP in the presence of IGF-1. +, present; −, absent.
- FIG. 7.
Ad-mediated overexpression of “kinase-dead” PKB blocks the protective effect of PHIP1 on FFA-induced INS-1 cell apoptosis. INS-1 cells were infected into six-well plates with AdGFP (MOI, 200), AdPHIP1 (MOI, 200), AdGFP (MOI, 200) plus AdKD (MOI, 600), or AdPHIP1 (MOI, 200) plus AdKD (MOI, 600). Sixteen hours postinfection, cells were incubated for 15 h with 15 mM glucose in the presence of 0.5% BSA alone or 0.4 mM OA-0.5% BSA. The incidence of apoptosis was assayed as described in Materials and Methods. Results are shown as the means ± SD of three independent experiments. *, P < 0.05 comparing results with AdPHIP1 to results with AdPHIP1 and AdKD in the absence of IGF-1. Lower panel, immunoblot analysis for PHIP1, phospho-Ser473 PKB (pSer473 PKB), and actin in cells treated as indicated above. The immunoblots are representative of two independent experiments. +, present; −, absent.
- FIG. 8.
Effect of PHIP1 on mRNA levels of PTEN, SHIP2, and mTOR in INS-1 cells. INS-1 cells were infected into six-well plates with AdGFP and AdPHIP1 (MOI, 400). Sixteen hours postinfection, cells were incubated for 15 h with 15 mM glucose in the presence of 0.5% BSA and collected for qPCR analysis. Experiments were performed three times in duplicate. Results are shown as the means ± SD.
