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Endocrinology, doi:10.1210/en.2007-0636
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Endocrinology Vol. 148, No. 11 5558-5565
Copyright © 2007 by The Endocrine Society

Pax4 Paired Domain Mediates Direct Protein Transduction into Mammalian Cells

Jun Lu, Ge Li, Michael S. Lan, Shuyu Zhang, Weiwei Fan, Hongwei Wang and Daru Lu

State Key Laboratory of Genetic Engineering (J.L., G.L., S.Z., W.F., D.L.), School of Life Science, University of Fudan, Shanghai 200433, China; and Department of Pediatrics (M.S.L., H.W.), Louisiana State University Health Sciences Center, the Research Institute for Children, Children’s Hospital, New Orleans, Louisiana 70118

Address all correspondence and requests for reprints to: Daru Lu, State Key Laboratory of Genetic Engineering, School of Life Science, University of Fudan, 220 Handan Road, Shanghai 200433, China. E-mail: drlu{at}fudan.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pax4, a paired-box transcription factor, is a key regulator of pancreatic islet cell growth and differentiation. Here, we report for the first time that the Pax4 protein can permeate into various cell types including pancreatic islets. The paired domain of Pax4 serves as a novel protein transduction domain (PTD). The Pax4 protein can transduce in a dose- and time-dependent manner. The cellular uptake of Pax4 PTD can be completely blocked by heparin, whereas cytochalasin D and amiloride were partially effective in blocking the Pax4 protein entry. Transduced intact Pax4 protein functions similarly to the endogenous Pax4. It inhibits the Pax6 mediated transactivation and protects Min6 cells against TNF{alpha}-induced apoptosis. These data suggest that Pax4 protein transduction could be a safe and valuable strategy for protecting islet cell growth in culture from apoptosis and promoting islet cell differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MEMBERS OF THE Pax transcription factor family that contains a paired domain are essential for the organogenesis and pluripotency of stem cell development (1). Nine members of the Pax gene family have been identified in mammalian genomes (Pax1–9). Pax4 and Pax6 share structural similarity containing a paired domain and a homeo-like domain but lack a conserved octapeptide (1). Both members were shown to be the key regulators in pancreatic islet differentiation (2, 3).

Genetic studies in humans have associated Pax4 gene mutations with type 2 diabetes and haplotypes with type 1 diabetes (4, 5, 6, 7). The functional role of Pax4 in β-cell development is still unclear. However, Pax4 expression in mice is initiated around embryonic d 9.5 in both pancreatic buds and becomes progressively restricted to β-cells until d 15. Mice lacking Pax4 are born normally but fail to develop any β- and {delta}-cells and rapidly develop severe diabetes as a consequence of hyperglycemia and dehydration (3, 8). Activin A (a member of the TGF-β family) stimulates Pax4 expression in pancreatic cell lines (9). Forced expression of Pax4 in rat and human islets produces a 3.5-fold increase in β-cell proliferation with an increase in oncogene c-myc and antiapoptotic gene Bcl-xL expression (10). These findings suggest that Pax4 plays a pivotal role in early pancreatic endocrine development and is operative during the late stage of β-cell differentiation and maturation. Furthermore, a recent report demonstrated that activation of Pax4 gene reprogrammed the PDX-1-VP-16-expressing cells into glucose-responsive mature insulin-producing cells (11).

Although gene transfer strategies are commonly used to elucidate gene function, the shortcomings of these approaches often are observed when using viral vehicles (12), and the gene copy number in transduced cells is unpredictable. Therefore, it is imperative to develop an alternative route of delivery such as transduction of proteins directly into living cells. Several reports have shown that small protein transduction domains (PTDs) from HIV/TAT protein (13), VP22 protein of Herpes simplex virus (14), and antennapedia homeoprotein of Drosophila (15) have the capabilities of penetrating cell membrane directly. Many PTD-fused full-length functional proteins have been demonstrated to transduce into cells and tissues (13, 16, 17, 18, 19, 20). Transcription factors involved in β-cell development were proven with PTDs. Pancreatic duodenal homeobox 1 (PDX1), a homeodomain protein, has its own antennapedia-like PTD. The PDX1 protein was successfully delivered into cultured islets and duct cells, and enhanced insulin gene expression was shown (21). BETA2/NeuroD and Ngn3 both contain a helix-loop-helix domain and were recently found to be transduced into cells recently (22, 23).

In the present study, we identified that a highly conserved paired domain of Pax4, rather than the homeo-like domain, is required to mediate Pax4 protein transduction into living cells. The transduced Pax4 protein functioned as effectively as endogenous Pax4 in pancreatic islets including inhibition of Pax6-mediated transactivation and prevention of islet cell apoptosis. Here, we report for the first time that Pax4 is capable of penetrating the cell membrane through the paired domain sequence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of Pax4 protein vectors and purification of recombinant Pax4 proteins
The mouse Pax4 cDNA was amplified by PCR using designed primers and subcloned into the EcoRI and XhoI sites of pET32a (Novagen, Madison, WI). The synthetic TAT sequence was subcloned into the BglII and NcoI sites of pET32a. Each amplified mutant sequences was subjected to sequence analysis to ensure their correct sequences. Pax5 and Pax6 paired domain fragments were amplified from pPax5.S1 (kindly provided by Dr. James Hagman, Integrated Department of Immunology, National Jewish Medical and Research Center, Denver, CO) and pcDNA3.0-Pax6 (kindly provided by Dr. Joachen Graw, Forschungszentrum für Umwelt und Gesundheit, the National Research Center for Environment and Health, Germany). The PCR products were subcloned into the EcoRI-HindIII sites of pET28a (Novagen) along with enhanced green fluorescent protein (EGFP) fragment (amplified by PCR from pEGFP-N2 and subcloned into the HindIII-NotI sites of pET28a). The resulting vectors were named as pPaired-A-EGFP, pPaired-B-EGFP, pPaired-EGFP, p132–171-EGFP, pHomeo-EGFP, p245–349-EGFP, p5Paired-A-EGFP, p5Paired-B-EGFP, p5Paired-EGFP, and p6Paired-EGFP. BL21 (DE3) cells containing the expression plasmid were grown at 37 C to an OD600 of 0.8. Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.7 mM, and the cells were incubated for 12 h at 25 C. Cells were sonicated, and the supernatants were recovered and applied to a column of Ni-nitrilotriacetic acid agarose (QIAGEN, Valencia, CA). The purified Pax4 protein was dissolved in buffer A [25 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM EDTA, and 150 mM imidazole].

Cell culture and protein internalization
HEK293 and HELF (human embryonic lung fibroblast) cells were cultured in DMEM containing 25 mM glucose, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. For Min6 cells, the culture medium also contained 5 µl/liter β-mercaptoethanol. All cells were cultured at 37 C in a 5% CO2 atmosphere. Rat pancreatic islets were isolated from male Sprague Dawley rats (provided by and bred in the Laboratory Animal Center of Second Military Medical University, China, certificate No. SCXK, 2002-011; 200–250 g) using collagenase (Roche Molecular Biochemicals, Indianapolis, IN) digestion and Histopaque-Ficoll (Sigma Chemical Co., St. Louis, MO) density gradient centrifugation. The newly isolated islets were maintained in RPMI 1640 medium containing 11.2 mM glucose and 10% wt/vol FBS. HEK293 and HELF cells were plated at appropriate density. After 24 h incubation, the medium was exchanged for fresh medium containing the appropriate concentration of purified proteins. After the purified proteins treatment, cells were washed three times with PBS and observed for fluorescence signals or analyzed by Western blot analysis.

Western blot analysis
Cell extracts were fractionated by 12% gel and transferred to polyvinylidene difluoride membranes (Immobilon-P PVDF Membrane; Millipore, Bedford, MA). Membranes were blocked at room temperature for 1 h in TBS/Tween 20 [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] with 5% nonfat dry milk. Membranes were then incubated overnight at 4 C in TBS/Tween 20 containing 5% nonfat dry milk and including either mouse anti-6-histidine antibody (1:5000; Invitrogen, Carlsbad, CA) or mouse anti-GAPDH antibody (1:5000; Sigma). Secondary antimouse IgG antibody coupled to horseradish peroxidase (1:2500; Promega, Madison, WI) was incubated at room temperature for 1 h and developed using a SuperSignal West Pico chemiluminescent kit (Pierce, Rockford, IL).

Treatment with amiloride, cytochalasin D, and heparin
To determine the mechanisms of Pax4 protein internalization, HELF cells were plated and cultured in DMEM with 10% FBS. Cells were pretreated for 30 min in serum-free medium with either 5 mM amiloride (Sigma), 10 µM cytochalasin D (Sigma), or 2 µg/ml heparin (Sigma). Pax4 PTD was added for 2 h in the presence of inhibitor, washed three times, and switched to complete medium for fluorescence examination.

Immunostaining
HELF cells were fixed with 4% paraformaldehyde, washed with PBS, and permeabilized with 1% Triton X-100 in PBS. Cells were blocked with blocking buffer (PBS, 1% Triton X-100, and 5% BSA) and incubated at 4 C with mouse anti-6-histidine (1:2500; Invitrogen) overnight. Rhodamine-conjugated goat antimouse was added for 45 min at room temperature (Jackson ImmunoResearch, West Grove, PA). Nuclear counterstaining was performed using 4,6-diamidino-2-phenylindole (DAPI) (Sigma).

EMSA
For this assay, transduced protein was tested for its ability to bind the pancreatic islet cell enhancer sequence (PISCES) sequences (AGGGGATGCA GTCAAGCGTG AATAATTGG) (24) using an EMSA (Pierce LightShift chemiluminescent EMSA kit). Nuclear extracts were prepared, and 20 µg protein was examined using biotin-labeled double-strand oligonucleotide probes. The reaction mixture was analyzed by electrophoresis in a nondenaturing 5% acrylamide gel with cold 0.5 Tris-borate-EDTA running buffer.

Luciferase reporter assay
The five copies of PISCES motif and the hsp70 mini-promoter acquired by PCR were constructed into pGL-3 Basic vector (Promega) between KpnI, XhoI, and HindIII sites to form the luciferase reporter plasmid pPISCES-luc. A dual luciferase assay was carried out according to manufacturer’s protocol (Promega). HELF cells at 50–60% confluence were transfected with pPISCES-luc, pcDNA3.0-Pax6, pRL-TK, and pcDNA3.0-Pax4 or with empty pcDNA3.0 as control vector with Lipofectamine 2000 (Invitrogen) on d 1. The cells were then cultured with 100 nM TAT protein, 100 nM Pax4 protein, or buffer A for an additional 24 h. The cells were harvested for luciferase activities using a luminometer (Luma LB9507; EG&G Berthold, Bad Wildbad, Germany). All experiments were performed in triplicate and repeated three times.

Microculture tetrazolium (MTT) assay
Cell viability was determined by a short-term MTT assay. In a 96-well microplate, 1 x 105 Min6 cells per well were incubated in 100 µl culture media and exposed to different concentrations of Pax4 protein or TAT protein as a control for 8 h. TNF{alpha} (12 ng/ml) was added for 12 h, and the media were replaced with 90 µl DMEM (containing no phenol red or FBS) and 10 µl MTT solution (5 mg/ml phosphate buffer) for 4 h. After the MTT-containing DMEM was removed, the remaining formazan blue crystals were dissolved in dimethylsulfoxide solution. Absorbance at 492 nm was measured using a microplate reader (model FL 311; Bio-Tek Instruments, Winooski, VT).

Quantitative RT-PCR
Total RNA from islet cells was extracted using Trizol reagent (Invitrogen), and cDNA was synthesized by RT using ReverTra Ace (Toyoba Chemical Co., Aichi, Japan). The primers for quantitative RT-PCR were rat Bcl-xL forward 5'-GTTGGATGGCCACCTATCTG-3' and reverse 5'-AAGAGTGAGCCCAGCAGAAC-3' and rat c-myc forward 5'-CTCCTCGCGTTATTTGAAGC-3' and reverse 5'-CAGCAGCTCGAATTTCTTCC-3'. The 10-µl reaction mixture contained 1 µg cDNA, 125 nM of both sense and antisense primers, and SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA) was used for PCR. Amplification and detection of specific products were performed with the ABI PRISM 7900 Sequence Detection System (PE Applied Biosystems). PCR cycle conditions were 95 C for 10 min followed by 40 cycles of denaturation at 95 C for 15 sec and annealing and extension at 60 C for 1 min. Reactions were performed in triplicate with rat β-actin as an internal control. Fluorescent signals were normalized to an internal reference ({Delta}Rn), and the threshold cycle (Ct) was set within the exponential phase of PCR. The relative gene expression is calculated by comparing cycle times for each target PCR. Cycle threshold values were converted to relative gene expression levels using the 2–({Delta}Ct sample–{Delta}Ct control)method as described elsewhere by Li et al. (25).

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay
Apoptosis was detected using the Deadend Fluorometric TUNEL System (Promega). Briefly, rat islets were seeded on chamber slides for 3 d and then exposed to purified Pax4 protein or buffer A for 4 d (protein added on d 0 and 3). After treatment, cells were fixed with 4% paraformaldehyde, permeabilized by 0.1% Triton X-100, and incubated with nucleotide mix and recombinant TdT enzyme mixture at 37 C for 1 h in the dark. The slides were counterstained with DAPI for 15 min on ice. Negative controls were obtained by omitting recombinant TdT enzyme and using the same volume of labeling solution. Positive controls were obtained by treating cells with DNase I (1000 U/ml for 30 min at 37 C) before labeling procedures. The data are expressed as the percentage of TUNEL-positive cells relative to total cell population in each group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transduction of intact Pax4 protein into cells
To test whether the purified Pax4 protein can be transduced into cells, HELF cells were cultured with 100 nM recombinant His-tagged Pax4 protein for 12 h. Immunostaining of His-tagged Pax4 protein revealed efficient transduction of intact recombinant Pax4 protein. Although the staining appears to be located mostly in cytoplasm, some of the rhodamine showed colocalization with nuclear staining (Fig. 1AGo, arrow). Furthermore, both the cytosolic extracts and nuclear extracts of HELF cells treated with Pax4 protein were detected by Western blot analysis using anti-6-histidine antibody (Fig. 1BGo). This result demonstrated that the intact Pax4 protein not only is capable of penetrating the cell membrane but also transduced into nuclei.


Figure 1
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  		FIG. 1. Transduction of intact Pax4 protein. A, HELF cells were treated with purified Pax4 protein (100 nM) for 12 h. Treated cells were immunostained by an anti-6-histidine and a rhodamine-conjugated secondary antibody. Nuclear staining (blue) was shown by DAPI. Arrows show the nuclear staining of Pax4 protein. B, Western blot analysis using anti-6-histidine (6His) antibody on cytosolic extracts and nuclear extracts of HELF cells treated with Pax4 protein.

 
Determining the PTD in Pax4 protein
To determine the PTD in Pax4 transcription factor, we constructed several EGFP-tagged mutant Pax4 proteins (Fig. 2Go, A and B). Each fusion protein was purified using a Ni-nitrilotriacetic acid column from a bacterial expression system as shown in Coomassie blue staining (Fig. 2CGo). Paired-EGFP and Paired-A-EGFP, but not the other fusion proteins, could be transduced into HELF cells. Paired-EGFP showed strong green fluorescence, whereas Paired-A-EGFP showed weak green fluorescence (Fig. 2DGo). Purified EGFP without PTD was used as a negative control (Fig. 2D1Go). The cytosolic and nuclear extracts of HELF cells treated with Paired-EGFP were also detected by Western blot analysis using an anti-6-histidine antibody (Fig. 2EGo). These data revealed that paired domain in Pax4 represents a unique PTD and is required for the Pax4 protein to be effectively transduced into cells.


Figure 2
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  		FIG. 2. Pax4 paired domain is a novel PTD. A, Structural diagram of the mouse Pax4 protein contains a paired domain (1–131 amino acids) of two conserved helix-turn-helix motifs and a homeodomain (172–229 amino acids). aa, Amino acids. B, A series of deleted Pax4 fragments were fused with EGFP (B2–B7); 6His-EGFP represents a control protein. Paired-A represents paired domain A of Pax4; Paired-B represents paired domain B of Pax4. C, Purified deleted Pax4 proteins were shown by Coomassie blue staining. Each fragment size is consistent with the predicted number of amino acids. D, HELF cells were treated with each deleted Pax4 fusion protein. Paired-EGFP showed strong fluorescence signal and Paired-A-EGFP showed weak fluorescence signal after 4 h treatment, whereas other fragments (B3, B5, B6, and B7) failed to show signal. E, Western blot analysis using anti-6-histidine (6His) antibody on cytosolic extracts and nuclear extracts of HELF cells treated with Paired-EGFP protein.

 
Transduction specificity of the Pax4 PTD
To test whether the purified Paired-EGFP protein can transduce various cell types, we examined HEK293, HELF, Min6, and primary rat islets for Paired-EGFP protein transduction. After 4 h of protein treatment, Paired-EGFP was observed as a strong fluorescence signal in HEK293, HELF, Min6, and primary islets cells (Fig. 3Go). The paired domain of Pax4 transcription factor is sufficient to direct proteins into four different cell types that were tested.


Figure 3
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  		FIG. 3. Transduction of Paired-EGFP protein into different cells. Four different cells, HEK293, HELF, Min6, and primary islets, were treated with Paired-EGFP protein for 4 h. All four cell types demonstrated strong fluorescence signal, indicating that Paired-EGFP internalized into cells readily.

 
Paired domains derived from other members of Pax family could be transduced into cells
To determine whether the paired domains derived from the other Pax proteins were able to facilitate protein transduction, Pax5 and Pax6 paired domains were chosen to be tested. Pax5 belongs to another subgroup of the Pax family that contains paired domain and the conserved octapeptide but has only an amino-terminal remnant homeodomain (Fig. 4AGo) (1). Fusion proteins 5Paired-EGFP, 5Paired-A-EGFP, 5Paired-B-EGFP, and 6Paired-EGFP were constructed and purified. HELF and HEK293 cells were incubated with each protein for 4 h. 5Paired-EGFP and 6Paired-EGFP fusion proteins could permeate both cells (Fig. 4Go, B1 and E). The EGFP signals of 5Paired-A-EGFP were weaker than that of 5Paired-EGFP protein, and 5Paired-B-EGFP did not show any fluorescence signal, which is consistent with the Pax4 results. We also extracted proteins from HELF cells for Western blot analysis (Fig. 4DGo). The internalized fusion protein levels are consistent with the intensity of green fluorescence. The protein loading in Western blot was normalized by GAPDH. Here, we demonstrated that the paired domains derived from different Pax proteins were capable of facilitating protein transduction, suggesting a common feature possessed by the other Pax proteins.


Figure 4
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  		FIG. 4. Transduction of Pax5 and Pax6 PTD. A, Structural diagram of the mouse Pax5 protein contains paired domain, the conserved octapeptide, and a partial homeodomain. B, HEK293 and HELF cells were treated with 500 nM EGFP-fused proteins (B1–B3) for 4 h; 5Paired-EGFP and 5Paired-A-EGFP could transduce into cells. C, Three deleted Pax5 were fused to EGFP (B1–B3); 5Paired represents paired domain of Pax5; 5Paired-A represents paired domain A of Pax5; 5Paired-B represents paired domain B of Pax5. D, Western blot analysis of cell extracts derived from HELF cells treated with EGFP-fused proteins. GAPDH expression was used as a loading control. 6His, Anti-6-histidine antibody. E, HEK293 and HELF cells were treated with 500 nM EGFP-fused 6Paired-EGFP protein for 4 h.

 
Dose- and time-dependent protein transduction of Pax4 PTD
To test the effectiveness of Pax4 PTD transduction, HELF cells were treated with Paired-EGFP protein at different concentrations for 4 h. The levels of transduced proteins were consistent with various dosages used to treat HELF cells (Fig. 5AGo). In addition, HELF cells were treated with 500 nM Paired-EGFP at various time points. The protein began to enter the cell after incubation for 5 min and reached maximal uptake within 4 h (Fig. 5BGo), consistent with a previous report about PDX-1 (26). Cell extracts of treated HELF cells were also tested by Western blot analysis (Fig. 5Go, C and D). These results suggested that Pax4 PTD could quickly and efficiently transduce into cells in a dose- and time-dependent manner.


Figure 5
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  		FIG. 5. Dose- and time-dependent transduction of Paired-EGFP protein. A, HELF cells were treated with Paired-EGFP at different concentrations. B, HELF cells were treated with 500 nM Paired-EGFP for 5 min to 4 h. C, Western blot analysis using anti-6-histidine (6His) and anti-GAPDH antibodies on extracts of HELF cells treated with Paired-EGFP protein at different concentrations. D, Western blot analysis on extracts of HELF cells treated with Paired-EGFP protein for 5 min to 4 h. GAPDH expression was used as a loading control. The same membrane was reprobed with the second antibody after stripping off the first signal.

 
Mechanisms of Pax4 PTD protein transduction
We further analyzed the molecular mechanisms of how the Pax4 PTD transduce into cells. It was reported that TAT PTD enters cells by macropinocytosis, which is a rapid, lipid raft-dependent, and receptor-independent form of endocytosis (16, 27). To examine whether macropinocytosis was also involved in Pax4 PTD translocation, HELF cells were preincubated in serum-free medium for half an hour with amiloride (a specific inhibitor of the Na+/H+ exchange required for macropinocytosis) (28) or cytochalasin D (an F-actin elongation inhibitor) (29). Pretreated cells were then incubated with Paired-EGFP for 2 h. After the treatment, both inhibitors reduce the intensity of green fluorescence, indicating less Paired-EGFP entering cells, but the inhibition is far from complete (Fig. 6Go, C and D). These observations indicated that macropinocytosis is partially involved in Pax4 PTD transduction.


Figure 6
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  		FIG. 6. Mechanisms of Pax4 PTD protein transduction. A, Control of Paired-EGFP entry into HELF cells. The cells were incubated in the presence of Paired-EGFP protein at 500 nM for 2 h. B, Cells were pretreated with 2 µg heparin, incubated with Paired-EGFP for 2 h. Heparin can significantly inhibit the Paired-EGFP internalization. C, Effect of 10 µM cytochalasin D on Paired-EGFP entry. Cells were pretreated with cytochalasin D and incubated with Paired-EGFP for 2 h. Fluorescence signal was reduced. D, Effect of 5 mM amiloride on Paired-EGFP entry. Cells were treated as above. Fluorescence signal was reduced.

 
Another potential mechanism is the adherence of PTDs to cell surface glycosaminoglycans (30, 31, 32). This adherence can be blocked by competitive binding of PTDs with heparan sulfate or heparin. To test whether heparin can block the adherence of Pax4 PTD to cell membrane, HELF cells were preincubated in serum-free medium for half an hour with heparin, and Paired-EGFP was then added into the medium. As shown in Fig. 6BGo, the EGFP signals were significantly decreased, suggesting the important roles of cell surface glycosaminoglycans during the internalization of the Pax4 PTD.

Transduced Pax4 protein maintains its functional effect as endogenous transcription factor
The PISCES motif is the binding sequence of Pax4 (24). We therefore used a gel shift assay to test whether the transduced Pax4 protein binds to the PISCES motif. HELF cells were treated with Pax4 protein, TAT protein, or buffer A for 12 h. The purified Pax4 protein was used as a positive control. The PISCES binding complex was observed in nuclear extracts from treated HELF cells. The specificity of the band was confirmed by the binding complex being decreased by unlabeled PISCES oligonucleotides (Fig. 7AGo).


Figure 7
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  		FIG. 7. Effects of transduced Pax4 on PISCES target site. A, HELF cells were treated with Pax4 (4 ) for 12 h, nuclear extracts from HELF cells were used in an EMSA. A shift band of PISCES (P) binding complex (arrow) was observed with nuclear extracts from treated HELF cells. Unlabeled PISCES (unP) were used to show specificity. The purified Pax4 protein was used as a positive control. The nuclear extracts from untreated and TAT protein (T) treated HELF cells were used as negative controls. This gel is representative of three independent experiments. B, Transduced Pax4 protein inhibits the Pax6-mediated transactivation of target promoter. HELF cells were transfected with pPISCES-luc, pcDNA3.0-Pax6, and pcDNA3.0 on d 1 and then incubated for 24 h with buffer A (C1), 100 nM TAT protein, and 100 nM Pax4 protein. C2 represents cells transfected with pPISCES-luc, pcDNA3.0-Pax6, and pcDNA3.0-Pax4 and then treated with buffer A. Transfection was repeated at least three times and normalized with internal control pRL-TK. *, P < 0.05; **, P < 0.01.

 
The PISCES motif is also the binding sequence of Pax6 (24). Pax4 inhibits Pax6-mediated transactivation by direct DNA binding site competition with Pax6 (33). To reveal whether the transduced Pax4 protein still maintain its functional effect as a transcription factor, we use Pax6 transactivation of pPISCES-luc reporter assay to access Pax4 protein competition. pcDNA3.0-Pax4 expression vector was used as a positive control. Transduced Pax4 protein suppressed 25% of the reporter activity (paired t test, P < 0.05; n = 3) as compared with 48% suppression by the pcDNA3.0-Pax4 vector (paired t test, P < 0.01; n = 3), whereas transduced TAT protein didn’t suppress the reporter activity (Fig. 7BGo). These results indicated that the internalized Pax4 protein could antagonize Pax6-induced transcription and maintain its functional effect after PTD-mediated transduction.

Pax4 protein prolongs islet cell survivor and protects against TNF{alpha}-induced apoptosis
Adenoviral-mediated overexpression of Pax4 in islets was shown to protect against cytokine-induced apoptosis (10). To examine whether transduced Pax4 protein can protect TNF{alpha}-induced apoptosis similar to the endogenous Pax4, the Pax4 protein-pretreated Min6 cells were exposed to TNF{alpha}. TAT protein treatment was used as a control. Cell viability was independently determined using the MTT assay. Treatment with TNF{alpha} induced 38% cell death (Fig. 8AGo, C2 and C1). Min6 cells pretreated with Pax4 protein (25–100 nM) enhanced cell viability to 96% as compared with the control cells (Fig. 8AGo). Transduction of the control protein TAT revealed a dose-dependent increase of protection, but the increment was not significant. The Pax4 transduction experiment revealed that the Pax4 protein can protect against apoptosis in a dose-dependent manner.


Figure 8
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  		FIG. 8. Pax4 protein protects against apoptosis. A, Effects of Pax4 protein on protection of the TNF{alpha}-induced apoptosis. Min6 cells were treated with Pax4 protein for 8 h and incubated with 12 ng/ml TNF{alpha} for an additional 12 h, and cell viability was measured using the MTT assay (percentage of cell viability). TAT protein was used as a control. C1 represents cells untreated with protein and TNF{alpha}; C2 represents cells treated with buffer A and TNF{alpha}. Cells were also treated with TNF{alpha} and different concentrations of Pax4 (25, 50, or 100 nM) or TAT protein. B, Rat islets were cultured with buffer A (C1), TAT protein, or Pax4 protein for 4 d. Cell death (percent) was measured by the TUNEL assay. C, Expression of Bcl-xL and c-myc in islet cells determined by quantitative RT-PCR. Islet cells were exposed to purified Pax4 protein or buffer A. C represents cells treated with buffer A; 41, 42, and 43 represent cells treated with 25, 50, and 100 nM Pax4, respectively. *, P < 0.05; **, P < 0.01.

 
To assess the impact of Pax4 on rat islet survival, TUNEL assay was performed to characterize the effect of Pax4 treatment on prolonging the Sprague Dawley rat islet survival in culture. As shown in Fig. 8BGo, treatment with Pax4 protein significantly attenuated apoptosis (7.8% TUNEL-positive cells) compared with 21% in buffer A and 19% in TAT protein-treated cultured islets (P < 0.01). Islet cells were exposed to purified Pax4 protein (25–100 nM) or buffer A for 4 d (protein added on d 0 and 3). We further tested whether Pax4 was able to enhance the expression of Bcl-xL and c-myc. Quantitative RT-PCR revealed a 2.88-, 3.01-, and 3.43-fold increase in Bcl-xL transcripts and a 1.60-, 1.78-, and 2.01-fold increase in c-myc transcripts in Pax4-treated cells (Fig. 8CGo). Together, our results suggest that transduced Pax4 protected islet cells against apoptosis through the activation of Bcl-xL, consistent with a previous report (10), and prolonged islet cell survival in culture.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proteins containing PTDs are capable of penetrating the cell membrane into cytoplasm and/or nuclei (21, 22, 23). In the present study, we identified a new PTD derived from the paired domain of members of the Pax gene family. The paired domain is a structural domain highly conserved through evolution that encodes a protein sequence with DNA-binding activity (Fig. 9AGo) (1). Our studies showed that the paired domain of the Pax family is sufficient to transport recombinant fusion protein or intact Pax4 protein into living cells. The transduced protein retained their biological functions as the endogenous Pax4 protein. This PTD also lacks cell type specificity as shown by four different cell types tested in our experiment. The Pax4 PTD transduction efficiency occurs in a dose- and time-dependent manner. These features are important for its application in cellular protein therapy. Both intact Pax4 and EGFP-tagged paired domain display highly efficient transport of exogenous proteins across the cell membrane, suggesting the paired domain alone can serve as a carrier sequence to transduce other foreign proteins. Although there is little information to compare the abilities of various PTDs to facilitate protein transport across cell membrane, paired domain derived from the Pax family clearly represents a novel PTD for direct protein delivery. Identification of more PTDs will definitively be beneficial for intracellular protein delivery and cell transplantation therapy.


Figure 9
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  		FIG. 9. Paired domain of Pax proteins. A, Multiple sequence alignment of paired domains among Pax4, Pax5, and Pax6. Basic amino acid residues are shown in light blue. Sequences and abbreviations are as follows: Pax4, Mus musculus (gi:2911811); Pax5, M. musculus (gi:6679213); and Pax6, M. musculus (gi:17227115). The multiple alignment was built using MegAlign and refined manually. B, Three-dimensional structure of Pax6 paired domain (Protein Data Bank accession code 6PAX). Basic amino acid residues are shown in blue. The three-dimensional picture was generated from 3D-Mol viewer (Invitrogen).

 
It was reported that a lysine- and arginine-rich peptide with 11–16 amino acids can permeate cells (34, 35). Other small PTDs, such as TAT PTD, a highly basic peptide, were shown to be quite effective for transporting proteins into cells (15, 34). But the paired domain of Pax4 consists of 128 amino acids with only 13% of basic amino acids distributed over the domain (Fig. 9AGo). Because the paired domain contains two conserved helix-turn-helix motifs, we separated the paired domain of Pax4 and Pax5 into part A and B. The EGFP signals of Paired-A-EGFP were much weaker than that of the Paired-EGFP protein treatment, whereas Paired-B-EGFP did not show any fluorescence signal. Thus, the intact paired domain possessed the cell penetration property. We speculate that the paired domain possessing the cell penetration property could be mediated through a highly conserved three-dimensional structure, where the arginine and/or lysine basic amino acids are situated on one side of the paired domain structure. We have generated a three-dimensional structure of Pax6 paired domain with conserved basic amino acids labeled in blue (Fig. 9BGo). It is still unclear which basic amino acids contribute to the membrane attachment and penetration. A more comprehensive mutagenesis study is required to elucidate the molecular events contributing to the Pax4 paired domain penetration. Although different PTDs share few sequence similarities, the molecular mechanisms of how the transduced proteins attach and penetrate cell membrane appear similar. Pax4 transduction can be completely blocked by heparin, whereas TAT PTD entered cells by macropinocytosis, which was also shared partially with Pax4 (16, 19, 27).

Pax4 is an important transcription factor for pancreatic endocrine cell development (3). It has been shown that Pax4 could promote islet cell proliferation and survival (10). The functional effects of Pax4 protein in driving endocrine precursor cells into islets and prolonging islet cells in culture raise an interesting application of using this unique PTD to deliver Pax4 protein in islet cell culture. Although the fluorescence staining of Pax4 protein was predominantly localized in the cytoplasm region of the transduced cells in contrast to the expected nuclei location, prolonged Pax4 protein treatment seems to increase the translocation of Pax4 protein into nuclei. The transduced Pax4 protein can inhibit Pax6-mediated transactivation and protect islet cells from apoptosis. Therefore, adding exogenous Pax4 proteins into the pancreatic precursors or islet cells presents a promising alternative approach to promote and maintain islet cell growth in culture. We have shown that BETA2/NeuroD and Ngn3 both can transduce by their basic helix-loop-helix domain (22). PDX-1 protein induces insulin expression in pancreatic duct cells (21). Pax4 protein can be used with these transcription factors and may facilitate islet progenitor cell differentiation into insulin-producing cells.


   Acknowledgments
 
We thank Matthias Austen (Director of Stem Cell Research, Germany) for pPax4, James Hagman (National Jewish Medical and Research Center, Denver, CO) for pPax5.S1, and Joachen Graw (Forschungszentrum für Umwelt und Gesundheit, the National Research Center for Environment and Health, Germany) for pcDNA3.0-Pax6. We thank Yuanfeng Wu and Dr. Guoyuan Liu (State Key Laboratory of Genetic Engineering, School of Life Science, University of Fudan, China) for their technical assistance.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online August 23, 2007

Abbreviations: DAPI, 4,6-Diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; MTT, microculture tetrazolium; PISCES, pancreatic islet cell enhancer sequence; PDX-1, pancreatic duodenal homeobox 1; PTD, protein transduction domain; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP nick end-labeling.

Received May 14, 2007.

Accepted for publication August 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chi N, Epstein JA 2002 Getting your Pax straight: Pax proteins in development and disease. Trends Genet 18:41–47[CrossRef][Medline]
  2. St-Onge L, Sosa-Pineda B, Chowdhury K, Mansouri A, Gruss P 1997 Pax6 Is required for differentiation of glucagon-producing {alpha}-cells in mouse pancreas. Nature 387:406–409[CrossRef][Medline]
  3. Sosa-Pineda B, Chowdhury K, Torres M, Oliver G, Gruss P 1997 The Pax4 gene is essential for differentiation of insulin-producing β-cells in the mammalian pancreas. Nature 386:399–402[CrossRef][Medline]
  4. Biason-Lauber A, Boehm B, Lang-Muritano M, Gauthier BR, Brun T, Wollheim CB, Schoenle EJ 2005 Association of childhood type 1 diabetes mellitus with a variant of PAX4: possible link to β-cell regenerative capacity. Diabetologia 48:900–905[CrossRef][Medline]
  5. Holm P, Rydlander B, Luthman H, Kockum I 2004 Interaction and association analysis of a type 1 diabetes susceptibility locus on chromosome 5q11–q13 and the 7q32 chromosomal region in Scandinavian families. Diabetes 53:1584–1591[Abstract/Free Full Text]
  6. Shimajiri Y, Sanke T, Furuta H, Hanabusa T, Nakagawa T, Fujitani Y, Kajimoto Y, Takasu N, Nanjo K 2001 A missense mutation of Pax4 gene (R121W) is associated with type 2 diabetes in Japanese. Diabetes 50:2864–2869[Abstract/Free Full Text]
  7. Shimajiri Y, Shimabukuro M, Tomoyose T, Yogi H, Komiya I, Takasu N 2003 PAX4 mutation (R121W) as a pirodiabetic variant in Okinawans. Biochem Bioph Res Co 302:342–344[CrossRef][Medline]
  8. Sosa-Pineda B 2004 The gene pax4 is an essential regulator of pancreatic β-cell development. Mol Cells 18:289–294[Medline]
  9. Ueda Y 2000 Activin A increases Pax4 gene expression in pancreatic β-cell lines. FEBS Lett 480:101–105[CrossRef][Medline]
  10. Brun T, Franklin I, St-Onge L, Biason-Louber A, Schoenle EJ, Wollheim CB, Gauthier BR 2004 The diabetes-linked transcription factor PAX4 promotes β-cell proliferation and survival in rat and human islets. J Cell Biol 167:1123–1135[Abstract/Free Full Text]
  11. Tang DQ, Cao LZ, Chou W, Shun L, Farag C, Atkinson MA, Li SW, Chang LJ, Yang LJ 2006 Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation. Lab Invest 86:829–841[Medline]
  12. Brunetti-Pierri N, Palmer DJ, Beaudet AL, Carey KD, Finegold M, Ng P 2004 Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates. Hum Gene Ther 15:35–46[CrossRef][Medline]
  13. Schwarze SR, Dowdy SF 2000 In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci 21:45–48[CrossRef][Medline]
  14. Elliott G, O’Hare P 1997 Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 88:223–233[CrossRef][Medline]
  15. Joliot A, Prochiantz A 2004 Transduction peptides: from technology to physiology. Nat Cell Biol 6:189–196[CrossRef][Medline]
  16. Wadia JS, Stan RV, Dowdy SF 2004 Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10:310–315[CrossRef][Medline]
  17. Embury J, Klein D, Pileggi A, Ribeiro M, Jayaraman S, Molano RD, Fraker C, Kenyon N, Ricordi C, Inverardi L, Pastori RL 2001 Proteins linked to a protein transduction domain efficiently transduce pancreatic islets. Diabetes 50:1706–1713[Abstract/Free Full Text]
  18. Kaplan IM, Wadia JS, Dowdy SF 2005 Cationic TAT peptide transduction domain enters cells by macropinocytosis. J Control Release 107:571–572[CrossRef]
  19. Lundberg M, Wikstrom S, Johansson M 2003 Cell surface adherence and endocytosis of protein transduction domains. Mol Ther 8:143–150[CrossRef][Medline]
  20. Richard JP, Melikov K, Brooks H, Prevot P, Lebleu B, Chernomordik LV 2005 Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J Biol Chem 280:15300–15306[Abstract/Free Full Text]
  21. Noguchi H, Kaneto H, Weir GC, Bonner-Weir S 2003 PDX-1 protein containing its own antennapedia-like protein transduction domain can transduce pancreatic duct and islet cells. Diabetes 52:1732–1737[Abstract/Free Full Text]
  22. Chen J, Li G, Lu J, Chen L, Huang Y, Wu HL, Zhang JX, Lu DR 2006 A novel type of PTD, common helix-loop-helix motif, could efficiently mediate protein transduction into mammalian cells. Biochem Biophys Res Commun 347:931–940[CrossRef][Medline]
  23. Noguchi H, Bonner-Weir S, Wei FY, Matsushita M, Matsumoto S 2005 BETA2/NeuroD protein can be transduced into cells due to an arginine- and lysine-rich sequence. Diabetes 54:2859–2866[Abstract/Free Full Text]
  24. Beimesche S, Neubauer A, Herzig S, Grzeskowiak R, Diedrich T, Cierny I, Scholz D, Alejel T, Knepel W 1999 Tissue-specific transcriptional activity of a pancreatic islet cell-specific enhancer sequence/Pax6-binding site determined in normal adult tissues in vivo using transgenic mice. Mol Endocrinol 13:718–728[Abstract/Free Full Text]
  25. Li H, Telemaque S, Miller RE, Marsh JD 2005 High glucose inhibits apoptosis induced by serum deprivation in vascular smooth muscle cells via upregulation of Bcl-2 and Bcl-xl. Diabetes 54:540–545[Abstract/Free Full Text]
  26. Noguchi H, Matsushita M, Matsumoto S, Lu YF, Matsui H, Bonner-Weir S 2005 Mechanism of PDX-1 protein transduction. Biochem Biophys Res Commun 332:68–74[CrossRef][Medline]
  27. Kaplan IM, Wadia JS, Dowdy SF 2005 Cationic TAT peptide transduction domain enters cells by macropinocytosis. J Control Release 102:247–253[CrossRef][Medline]
  28. West MA, Bretscher MS, Watts C 1989 Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J Cell Biol 109:2731–2739[Abstract/Free Full Text]
  29. Sampath P, Pollard TD 1991 Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments. Biochemistry 30:1973–1980[CrossRef][Medline]
  30. Console S, Marty C, Garcia-Echeverria C, Schwendener R, Ballmer-Hofer K 2003 Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J Biol Chem 278:35109–35114[Abstract/Free Full Text]
  31. Noguchi H, Ueda M, Matsumoto S, Kobayashi N, Hayashi S 2007 BETA2/NeuroD protein transduction requires cell surface heparan sulfate proteoglycans. Hum Gene Ther 18:10–17[CrossRef][Medline]
  32. Ziegler A, Nervi P, Durrenberger M, Seelig J 2005 The cationic cell-penetrating peptide CPP (TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence. Biochemistry 44:138–148[CrossRef][Medline]
  33. Ritz-Laser B, Estreicher A, Gauthier BR, Mamin A, Edlund H, Philippe J 2002 The pancreatic β-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6. Diabetologia 45:97–107[CrossRef][Medline]
  34. Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, Sugiura Y 2001 Arginine-rich peptides: an abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem 276:5836–5840[Abstract/Free Full Text]
  35. Pastori RL, Klein D, Ribeiro MM, Ricordi C 2004 Delivery of proteins and peptides into live cells by means of protein transduction domains: potential application to organ and cell transplantation. Transplantation 77:1627–1631[CrossRef][Medline]



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