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Endocrinology, doi:10.1210/en.2007-0127
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Endocrinology Vol. 148, No. 12 5874-5883
Copyright © 2007 by The Endocrine Society

Insulin-Increased Prolactin Gene Expression Requires Actin Treadmilling: Potential Role for P21 Activated Kinase

Frederick M. Stanley

Department of Pharmacology and NYU Cancer Institute, New York University School of Medicine, New York, New York 10016

Address all correspondence and requests for reprints to: Dr. Frederick M. Stanley, Department of Pharmacology, MSB407, New York University Medical Center, 550 First Avenue, New York, New York 10016. E-mail: stanlf01{at}med.nyu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-increased prolactin gene transcription in GH4 cells was enhanced by binding on fibronectin. This was mediated by receptor-like protein tyrosine phosphatase {alpha}, which activated Src, Rho, and phosphatidylinositol 3-kinase. It suggested that insulin signaling to gene transcription was partly dependent on actin rearrangement. This was confirmed through studies using inhibitors of actin treadmilling. Cytochalasin D, jasplakinolide, latrunculin B, and swinholide A altered the actin cytoskeleton of GH4 cells, as assessed by Alexa Fluor phalloidin staining, and inhibited insulin-increased prolactin gene transcription. These reagents did not affect the controls. Nor was it due to a gross defect of insulin signaling because activation/translocation of glycogen synthase kinase 3ß and mammalian target of rapamycin were not affected. Expression of wild-type and mutant actin treadmilling agents, Cdc42, TC10, neuronal Wiskott-Aldrich syndrome protein, and Nck, indicated that they were essential to insulin-increased prolactin gene expression, and suggested that activation of p21 associated kinase (PAK) might also be essential to this process. PAK expression also increased and PAK mutants decreased prolactin promoter activity in insulin-treated cells. The activation of PAK in the presence of inhibitors was also consistent with a role in activation of insulin-increased prolactin gene expression. Finally, small interfering RNA-mediated reduction of PAK decreased the effect of insulin on prolactin gene expression. Thus, it is likely that insulin activation of actin treadmilling through Cdc42/TC10 and neuronal Wiskott-Aldrich syndrome protein activates PAK and prolactin gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN BINDING TO its receptor activates the receptor’s kinase activity and increases substrate phosphorylation. The phosphorylated substrate, insulin-receptor substrate (IRS) and/or Shc, acts as an adapter that recruits and activates downstream signaling molecules, such as phosphatidylinositol (PI) 3-kinase. PI 3-kinase is activated when its p85 subunit interacts with phosphorylated IRS (1, 2). PI 3-kinase was required for insulin-increased prolactin gene transcription (3). PI 3-kinase phosphorylation of PI at the 3' position results in the activation of a number of kinases such as phosphoinositide-dependent protein kinase-1 and protein kinase B (Akt), whose substrates are not yet fully defined but include serum and glucocorticoid stimulated kinase, glycogen synthase kinase (GSK), and ribosomal protein S6 kinase (4). In addition, the kinase mammalian target of rapamycin (mTOR) is activated indirectly through phosphorylation and inhibition of the phosphatases tuberous sclerosis complex 1 and 2 by phosphoinositide-dependent protein kinase-1. These kinases mediate the effects of insulin. However, PI 3-kinase was not sufficient for all of the effects of insulin because activation of downstream effects of PI 3-kinase with a soluble PI-(3,4,5)-triphosphate could not fully recreate the effect of insulin (5). Recently, insulin was shown to activate through TC10, a Rho-related GTPase (6). Farnesylation and palmitoylation of TC10 colocalize it with the insulin receptor in lipid rafts. Steps downstream of TC10 have not yet been defined but are thought to involve actin rearrangement. This hypothesis follows from the observation that glucose transporter 4 containing microsomes are associated with and may be tethered to the actin cytoskeleton (7).

The actin cytoskeleton is in constant flux, which is termed treadmilling, because actin filaments (F-actin) are constantly being assembled from the barbed ends and disassembled from the pointed ends of the filaments (8, 9). This process involves a large number of proteins. Profilin binds to ADP-actin and accelerates ADP/ATP exchange. This speeds actin assembly into filaments. Cofilin severs F-actin at the pointed end of the filament and releases G actin. Cofilin is inactivated by phosphorylation at serine 3, which is mediated by Lim kinase. Lim kinase is itself activated through phosphorylation by Rho-dependent kinases (Rock). Rho can slow filament disassembly by activation of Rock. Gelsolin (capping protein) caps the barbed ends and inhibits further elongation of the filament.

The role of Rho-related GTPases in actin treadmilling is to localize the site and type of filaments that are formed (10). Rho is required for formation of stress fibers, Rac is activated at the site of lamellipodia formation, and Cdc42 is responsible for extension of filopodia. The formation of lamellipodia and filopodia involves new filament construction, whereas stress fibers depend on reorganization of existing filaments. The sequence of events was worked out for Cdc42 (11). Activated Cdc42 activates neuronal Wiskott-Aldrich syndrome protein (N-WASP). The Arp2/3 complex is then recruited to the membrane-associated N-WASP. This nucleates filament formation at distinct sites. Activation of N-WASP also unveils binding sites for other proteins such as Nck, which can recruit p21 associated kinase (PAK) to the complex for activation.

Interference with actin treadmilling affected the activation of a subset of genes (12). Inhibitors of actin treadmilling blocked serum activated induction of serum response factor (SRF) dependent promoters, and actin treadmilling was sufficient to explain the differential effect of serum on these promoters. This resulted from nuclear accumulation of MAL, an SRF coactivator, in response to actin treadmilling (13). Other mechanisms are also possible. Rearrangement of the actin cytoskeleton affects the activity of a number of enzymes such as PI 3-kinase and PAK, which could activate the transcription of some genes (14, 15). Actin-like proteins are also components of mammalian chromatin remodeling complexes, and actin activation of these complexes could explain the effects of actin-altering drugs on gene transcription (16).

These studies present evidence that insulin-increased prolactin gene expression depends on actin treadmilling/cytoskeletal reorganization. Inhibition of Cdc42 or TC10 blocked insulin-increased gene transcription. Inhibitors that caused aberrant development of the actin cytoskeleton also specifically blocked insulin-increased prolactin gene expression. This was not due to gross disruption of insulin signaling because insulin- and cAMP-increased phosphorylation and distribution of mTOR and GSK3ß were not affected. Expression of wild-type (WT) and mutant forms of N-WASP, and Nck{alpha}, also specifically affected insulin-increased prolactin gene expression. This suggested that PAK activated insulin-increased prolactin gene expression because Nck{alpha} recruits PAK to WASP. Expression of WT and mutant PAK and knockdown of PAK with small interfering RNA (siRNA) strongly support a role for PAK in insulin-increased prolactin promoter expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM containing 4.5 g/liter glucose and iron-supplemented calf serum was obtained from HyClone Laboratories (Logan, UT). T47D cells were obtained from the American Type Culture Collection (Manassas, VA). LY294002 and U0126 were from Calbiochem (San Diego, CA). Cytochalasin D and latrunculin B were from Calbiochem. Swinholide A and jasplakinolide were from Alexis Biochemicals (San Diego, CA). Alexa Fluor phalloidin was from Molecular Probes, Inc. (Eugene, OR). Reagents for assay of luciferase (Luc) and ß-galactosidase were from Promega Corp. (Madison, WI). All other reagents were of the highest purity available and were obtained from Sigma-Aldrich (St. Louis, MO), Pierce (Rockford, IL), Calbiochem, Bio-Rad Laboratories (Hercules, CA), Eastman Chemical Co. (Kingsport, TN), Thermo Fisher Scientific Inc. (Waltham, MA), or Roche Molecular Biochemicals (Indianapolis, IN).

Antibodies
Antibody against human influenza virus hemagglutinin was purchased from Covance (Richmond, CA). Anti-myc antibody was a gift from Dr. J. Sap (New York University, New York, NY). Anti-GFP was from Molecular Probes. Anti-Flag was purchased from Sigma-Aldrich. Anti-phospho mTOR serine 2442, phospho PAK serine 199, phospho PAK Ser 423, phospho Akt serine 473, anti-phospho GSK3ß serine 473, and anti-GSK3ß were from Cell Signaling Technology, Inc. (Beverly, MA). Anti-PAK and anti-Akt were from BD PharMingen (San Diego, CA). Anti ERK1 and anti ERK2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase linked secondary antibodies were from Pierce.

Plasmids
The construction of reporter plasmids containing –173/+75 of prolactin 5'-flanking DNA and Luc cDNAs was described previously (17, 18, 19). The human insulin expression vector, pRT3HIR2, was the gift of Dr. J. Whittaker (Case Western Reserve, Cleveland, OH) (20), and RSV-ß-Gal is from Dr. H. Samuels (New York University School of Medicine, New York, NY) (21). Myc tagged cdc42 N17 was the gift of Dr. A. Hall (University College, London, UK) and has been described previously (22, 23, 24). Vectors that expressed enhanced green fluorescent protein tagged N-WASP WT, the N-WASP mutants N-WASP{Delta}WH1 and N-WASP{Delta}WA were from Dr. M. Way (Cancer Research United Kingdom, Lincoln’s Inn Fields Laboratories, London, UK) (5). GFP-TC10 N31 was a gift from Dr. M. Czech (University of Massachusetts, Wooster, MA). Myc tagged PAK and PAK mutants were from Dr. J. Field (University of Pennsylvania School of Medicine, Philadelphia, PA) (25). HA tagged Nck{alpha} and SH2 and SH3 mutants were from Dr. Wei Li (University of Southern California, Los Angeles, CA) (26).

Analysis of prolactin promoter responsiveness using transient transfection
Electroporation experiments and reporter assays were performed as described (27). GH4 cells were harvested with an EDTA solution, and 6 x 106 cells were used for each electroporation. Trypan blue exclusion before electroporation ranged from 95–99%. The voltage of the electroporation was 1550 V. This gives trypan blue exclusion of 70–80% after electroporation. The transformed cells were plated in 96-well dishes (Falcon Plastics, Brookings, SD) at 1 x 105 cells per well in DMEM with 10% hormone-depleted serum. The cells were allowed to attach, and hormones were added for 24 h. The medium was replaced with assay buffer, and the plates were frozen at –80 C. Luc assays were performed using reagents and protocols from Promega.

Control of transfection efficiency was performed using an RSV-ß-galactosidase expression plasmid (0.5 µg/electroporation) (21). The ß-galactosidase activity in the cell lysates was determined using ß-Glo (Promega). Transfection efficiency did not vary significantly among transfections performed at the same time. The relative light units from the Luc assay were corrected for minor variations in ß-galactosidase activity by converting them to Luc relative light units/ß-galactosidase relative light units/mg protein. The fold stimulation or inhibition was then determined.

Western immunoblot analysis
For analysis of plasmid expression.
GH4 cells were harvested in a lysis buffer consisting of 50 mM HEPES (pH 7.5), 1% Triton X-100, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1 mM Na3VO4, 50 mM Na4P2O7, 1 mM NaF, 1 mM [4-(2-aminoethyl)-benzenesulfonylfluoride, HCl], and 10 µg/ml aprotinin. Protein was determined using the Bradford reagent (Bio-Rad) and analyzed by SDS-PAGE using 10% gels. The proteins were then transferred to nitrocellulose membranes (Micron Separations, Westborough, MA) and immunoblotted using enhanced chemiluminescence.

For kinase activation studies.
GH4 cells were harvested in an hypotonic buffer consisting of 10 mM HEPES (pH 7.5), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM Na3VO4, 1 mM (NH4)6Mo7O24, 10 mM NaF, 10 mM NaP2O7, 1 mM dithiothreitol, 1 mM AEBSF, and 10 µg/ml aprotinin. They were allowed to swell on ice for 10 min and lysed by addition of NP40 to a final concentration of 0.5%. The nuclei were collected by centrifugation for 1 min at 4 C and washed once. They were then extracted with a buffer containing 20 mM HEPES (pH 7.5), 0.5 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM (NH4)6Mo7O24, 10 mM NaF, 10 mM NaP2O7, 1 mM dithiothreitol, 1 mM AEBSF, and 10 µg/ml aprotinin. The samples were vortex for 15 sec at the highest setting and then placed on ice. The process was repeated four times. The nuclear extract was collected after a 10-min centrifugation at 14,000 x g. Protein was assayed and analyzed as previously described.

Cell staining and fluorescence microscopy
GH4 cells were inoculated onto poly-L-lysine treated coverslips at a density of 250,000 cells per coverslip. The media were exchanged at 24 h, and the cells were incubated an additional 24 h with various inhibitors of actin treadmilling. The coverslips were then washed twice with PBS. They were fixed using 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. The cells were then stained with Alexa Fluor 350 phalloidin (Molecular Probes) and mounted using Mowiol. The slides were photographed using a Zeiss Axio fluorescence microscope and Openlab software (Carl Zeiss, Inc., Thornwood, NY). Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) was used for post processing.

siRNA knockdown of PAK
The sequences of the three PAK isozymes from the rat and human were compared with find regions of similarity. A region in the CRB domain aa 119–125 of rPAK1 and 114–120 of rPAK3 was identical at the DNA level. There were two mismatches with the rat PAK2 and human PAK1/3 sequences. A sense siRNA, CCCACAGGCUGUUCUGGAUtt, and an antisense, AUCCAGAACAGCCUGUGGGtt sequence were synthesized and annealed (Ambion, Austin, TX). GH4 and T47D cells were inoculated into 96-well plates at 20,000 cells per well in growth medium. The cultures were treated with 10 nM siRNA to rPAK or a scrambled RNA using HiPerfect (QIAGEN, Inc., Valencia, CA), or were left untreated as a control. The next day the medium was exchanged for medium that contained charcoal-treated serum, and the treatment with siRNA was repeated. Three hours after refeeding, the cultures were transfected with Prl-Luc/ß-galactosidase using Genefector (Venn-Nova, Pompano Beach, FL) for the GH4 cells and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for the T47D cells. Insulin or forskolin was added 3 h after transfection, and the cells were incubated an additional 18–20 h.

Statistical analysis
InStat from GraphPad Software (San Diego, CA) was used for all statistical analysis. The data were subjected to one-way ANOVA, and the Tukey-Kramer multiple comparisons test was used to determine the significance of the observed differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Disorganization of the actin cytoskeleton blocks insulin-increased prolactin gene transcription
Activated mutants of Src and Rho increased basal and insulin-increased prolactin gene expression, and caused rearrangement of the cytoskeleton and morphology of GH4 cells (1). This suggested that cytoskeletal reorganization might be important for insulin-increased prolactin gene expression. Insulin causes increased prolactin secretion from GH4 cells, and secretion is a process that requires cytoskeletal alteration. Coupling of secretion with transcription would ensure that sufficient stores of prolactin were available when required. Thus, a common signal to activate both of these processes might be expected.

GH4 cells that were transfected with the prolactin-Luc reporter plasmid were treated with cytochalasin D, jasplakinolide, latrunculin B, or swinholide A for 2 h before addition of insulin for 24 h (Fig. 1Go). Cytochalasin D (Fig. 1AGo), jasplakinolide (Fig. 1BGo), latrunculin B (Fig. 1CGo), and swinholide A (Fig. 1DGo) blocked the effect of insulin on prolactin gene expression (P < 0.001), whereas cAMP-increased prolactin-Luc expression is not significantly affected (P > 0.05). T3-increased GH-Luc expression is likewise unaffected by these drugs (P > 0.05) (Fig. 1Go). This response was not unique to GH4 cells because these inhibitors also significantly reduced insulin-increased prolactin gene expression in T47D, a human breast cancer cell line (P < 0.001). The cAMP response in this cell line was also inhibited by treadmilling inhibitors (P < 0.05), whereas the T3 response of the GH Luc promoter remained insensitive (P > 0.05).


Figure 1
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  		FIG. 1. Disorganization of the actin cytoskeleton blocks insulin-increased prolactin gene transcription. GH4 cells, transfected with 10 µg Prl(–173/+75)Luc (insulin and cAMP) or 10 µg GH(–236/+6)Luc (T3), were incubated with the indicated concentrations of cytochalasin D (CD) (panel A), jasplakinolide (Jasp) (panel B), latrunculin B (LB) (panel C), or swinholide A (SA) (panel D) for 2 h, or were left untreated as controls. The prolactin-Luc transfected cultures were then treated for an additional 20–24 h with 1 µg insulin or 0.5 mM 8-CPT-cAMP (left axis). The GH-Luc transfected cultures were treated with 100 nM T3 (right axis). The normalized Luc expression in experimental cultures was compared with the level in untreated vector controls to determine the fold-stimulation (Fold-Basal). The average + SEM from three separate experiments done in triplicate is shown. T47D cells (panel E) were transfected with prolactin-Luc and 5 µg RSV-Pit1, and treated for 2 h with 2 µM cytochalasin D, 150 nM jasplakinolide, 2 µM Latrunculin B, or 40 nM swinholide A. They were then treated with insulin or cAMP for 20 h, and Luc activity was assayed as described previously.

 
To determine the extent of cytoskeletal disruption by these drugs, GH4 cells were plated on coverslips and treated with the various inhibitors for 24 h. They were then stained with Alexa Fluor phalloidin to visualize F-actin. The cortical actin ring seen in the control cells (Fig. 2Go) was disrupted by cytochalasin D, jasplakinolide, latrunculin B, and swinholide A. These inhibitors redistribute actin into intracellular compartment (cytochalasin D and jasplakinolide) or concentrated peripheral foci (swinholide A). Latrunculin B-treated cells also showed significant modification in actin distribution with most cells containing one to a few peripheral foci. Thus, treatments that interfere with the cortical actin ring of the GH4 cells inhibit insulin-increased prolactin gene transcription.


Figure 2
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  		FIG. 2. Inhibitors of actin treadmilling disorganize the cortical actin skeleton of GH4 cells. GH4 cells were cultured on coverslips and incubated with 1 µM cytochalasin D, 1 µM latrunculin B, 100 nM jasplakinolide, or 20 nM swinholide A for 24 h, or were left untreated as controls. They were stained with Alexa Fluor 350 phalloidin and photographed. Typical individual cells are shown.

 
Insulin signaling is not grossly affected by inhibitors of actin treadmilling.
A possible explanation for the effect of these inhibitors is that they disrupt insulin signaling to the nucleus. Insulin-increased prolactin gene expression in GH4 cells is wortmannin/LY294002 sensitive (30). This implies that insulin receptor activation results in the activation of Akt/protein kinase B, and its downstream targets such as GSK3ß and mTOR. Therefore, activation state-specific antibodies were used to determine the integrity of this pathway. GH4 cells were incubated for 24 h with the inhibitors and then incubated for 10 min with insulin or cAMP. Cytoplasmic and nuclear extracts were prepared, analyzed by SDS-PAGE, and blotted to nitrocellulose. The blots were probed with antibodies to GSK3ß phosphorylated at serine 9 and mTOR phosphorylated at serine 2448. These sites are phosphorylated in response to PI 3-kinase/Akt activation. As seen in Fig. 3Go, insulin and cAMP increase phosphorylation of these kinases. There were small effects on basal levels of kinase phosphorylation, but none of the inhibitors prevented increased phosphorylation in response to insulin in either the cytoplasm or nucleus. This implies that there was no gross disruption of insulin signaling by the inhibitors. It remained possible that a specific kinase mediating insulin-increased prolactin gene expression was not phosphorylated or translocated into the nucleus in response to insulin under these conditions.


Figure 3
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  		FIG. 3. Inhibitors of actin treadmilling do not affect insulin activation of insulin dependent kinases. GH4 cells were incubated for 24 h with 2 µM cytochalasin D, 150 nM jasplakinolide, 2 µM latrunculin B, or 40 nM swinholide, or were untreated as controls. Cytosolic (top) and nuclear (bottom) proteins were then prepared and analyzed by SDS-PAGE. The blots were sequentially probed with antibodies to GSK3ß phosphorylated on Ser9 (Cell Signaling Technology) or mTOR phosphorylated on Ser2448 (Cell Signaling Technology). The blots were probed with Erk 1/2 as a loading control. Use of Erk as a loading control allowed the use of the same probe for both nuclear and cytoplasmic blots.

 
Cdc42/TC10 was required for insulin-increased prolactin gene expression.
We next tested if Rho-related GTPases were required for insulin-increased prolactin gene expression because Rho-related GTPases direct the type and location of cytoskeletal rearrangement. Mutants of Rho and Rac that act as dominant negatives, Rho N19 and Rac N17, did not specifically reduce insulin-increased or basal prolactin gene transcription, even at high levels (data not shown). Transfection with small amounts of Cdc42 N17 and TC10 N31 prevented the increase in prolactin gene transcription in insulin-treated cells (Fig. 4AGo, Top; P < 0.001). The thyroid hormone (T3) response of the GH promoter served as a control for these experiments (Fig. 4AGo, middle). GH-Luc reporter activity was increased 3-fold by T3 in GH4 cells. This was not affected by any of the interfering Rho mutants. The expression of the Rho-related protein is shown in Fig. 4AGo, Bottom, and the activity of the cytomegalovirus (CMV) promoter was not affected by T3, insulin, or cAMP (data not shown).


Figure 4
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  		FIG. 4. Expression of mutant treadmilling proteins affects insulin-increased prolactin gene expression. Each panel consists of three sections. The top graph shows GH4 cells that were cotransfected with 10 µg Prl(–173/+75)Luc, 5 µg human insulin receptor, and 0.5 µg pRSV-ß-gal with expression vectors for various treadmilling proteins listed in A–D. The cultures were treated with 1 µg/ml insulin (shaded bars), 0.5 mM 8-CT-cAMP (white bars), or untreated controls (black bars) for 24 h. The normalized Luc expression in experimental cultures was compared with the level in untreated vector controls to determine the fold-stimulation (Fold-Basal). The average + SEM from three separate experiments done in triplicate is shown. The middle section of each panel represents GH4 cells that were cotransfected with 10 µg GH(–236/+6)Luc, 5 µg human insulin receptor, 0.5 µg pRSV-ß-gal with expression vectors for various treadmilling proteins described in A–D. The cultures were treated with 1 µM T3 for 24 h. The normalized Luc expression in experimental cultures was compared with the level in untreated vector controls to determine the fold-stimulation (Fold-Basal). The average + SEM from three separate experiments done in triplicate is shown. The bottom section of each panel shows Western blots of the expressed treadmilling proteins. A, Cotransfections included 1 µg Cdc42 N17, 1 µg TC10 N31, or 1 µg vector. The expression of N17 Cdc42 and N31 TC10 is shown at the bottom. B, Cotransfections included 10 µg N-WASP WT, 10 µg N-WASP WA, 10 µg N-WASP WH1, or 10 µg vector control. The expression of N-WASP proteins is shown at the bottom. C, Cotransfections included 1 µg Nck{alpha} WT, 1 µg Nck{alpha} SH2, 1 µg Nck{alpha} SH3, or 1 µg vector. The expression of Nck{alpha} proteins in the transfections is shown at the bottom. D, Cotransfections included 1 µg PAK WT, 1 µg PAK LL, 1 µg PAK KR, 1 µg PAK LLKR, or 1 µg vector. The expression of the PAK proteins in the transfections is shown at the bottom.

 
N-WASP is involved in insulin-increased prolactin-Luc expression.
Rho-related GTPases direct actin polymerization at specific sites in the cell. This requires that they assemble factors that can nucleate actin polymerization and branching. Ultimately, this means the Arp2/3 complex. Cdc42 and TC10 recruit Arp2/3 by activating N-WASP that has sites for binding of Arp2/3 as well as profilin/actin binding sites. Rac interacts with SCAR/WAVE to recruit Arp2/3, whereas Rho alters actin stress fibers through activation of ROCK and Lim kinases, and by reversing the auto-inhibition of mDia (31). Expression of N-WASP and N-WASP mutants would be expected to affect insulin-increased prolactin gene expression if insulin acted through Cdc42/TC10 rather than through another Rho-GTPase. Cotransfection of GH4 cells with vectors expressing WT and mutant N-WASP affected insulin-increased prolactin gene transcription (Fig. 4BGo, top). The WT N-WASP significantly increased basal prolactin gene transcription 10-fold, and insulin- and cAMP-increased prolactin-Luc expression approximately 5-fold (P < 0.001). The vector WASP-{Delta}WA expressed N-WASP with a deletion of the C-terminal WA domain containing verprolin and cofilin homology regions and the acidic region (verprolin cofilin acidic). This portion of the molecule binds actin and arp2/3 proteins. N-WASP without this region is not functional. The lack of a significant effect of this protein on insulin- or cAMP-increased prolactin-Luc expression (P > 0.05) indicated that the VCA domain and by extension, actin polymerization, was necessary for the effect of N-WASP stimulation. Finally, expression of N-WASP with an N-terminal deletion of the WH domain (WASP {Delta}WH1) blocked insulin- and cAMP-increased prolactin-Luc expression (P < 0.001). The N-terminal region is necessary for proper membrane localization of the N-WASP. T3 increased GH-Luc activity 3-fold, and this was not affected by expression of N-WASP WT or mutants (Fig. 4BGo, middle).

Nck and PAK affect insulin-increased prolactin gene expression.
Activation of N-WASP by Cdc42 or TC10 causes conformational changes that allow the nucleation of actin filaments by binding of Arp2/3 complex, but it also unveils sites for binding of the adaptor protein Nck, which can recruit other proteins to the N-WASP complex for activation. Expression of WT Nck{alpha} significantly increased insulin-increased prolactin gene expression (P < 0.01; Fig. 4CGo, top). It did not have a significant effect on cAMP-increased prolactin-Luc expression (P > 0.05). Mutants of the SH3 or SH2 domains of Nck{alpha} inhibited insulin-increased prolactin gene expression (P < 0.001). cAMP-increased prolactin Luc expression was not affected by these mutants. WT Nck{alpha} was the only treadmilling-related factor to significantly augment T3-increased GH Luc expression (P < 0.01). However, the mutants of Nck{alpha} had no effect on T3-increased GH Luc expression. This argues against direct involvement of Nck{alpha} in T3 signaling.

PAK is recruited to N-WASP by Nck{alpha} (32) and can also be activated by Cdc42/TC10. PAK activation by insulin-increased actin treadmilling could provide a link to insulin’s effect on the prolactin promoter. Expression of WT PAK significantly increased insulin- and cAMP-increased prolactin Luc expression (P < 0.001; Fig. 4DGo, top) but did not affect the GH promoter activated by T3 (Fig. 4DGo, middle). Expression of PAK mutants blocked insulin-increased prolactin Luc expression, whereas they did not significantly interfere with the effect of cAMP or the T3-increased GH Luc activity. PAK K299R expresses PAK with no kinase activity, whereas PAK LL has a mutation of the Rac/Cdc42 binding site that makes it p21 independent, and PAK LLKR is a double mutant. Each of the single mutants inhibit the effect of insulin more than 50% (P < 0.01), whereas the double mutant inhibits more than 80% (P < 0.001).

A kinase that mediated insulin responses would be expected to respond to insulin and inhibitors of insulin action in a way consistent with their effects on prolactin Luc expression. PAK is activated by insulin (Fig. 5Go), and insulin-activated PAK is found in both the cytoplasmic and nuclear fraction. This suggested that PAK could directly phosphorylate nuclear targets such as Elk-1/Sap1, which previously mediated insulin-increased prolactin gene transcription (30). Insulin-increased prolactin promoter expression was previously shown to be PI 3-kinase dependent because it was inhibited by LY294002 and Erk independent because PD98059, a MAPK kinase 1/2 inhibitor, stimulated insulin-increased prolactin expression. Likewise, PAK activation by insulin was inhibited by LY294002 but stimulated by the inhibitor U0126 (Fig. 5Go), which blocks MAPK kinase 1/2 more effectively than PD98059.


Figure 5
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  		FIG. 5. PAK activation by insulin is PI 3-kinase dependent. GH4 cells were incubated with 5 µM U0126 or 10 µM LY294002 for 2 h. These inhibitor concentrations were previously shown to inhibit the effects of cAMP (U0126) and insulin (LY294002) on prolactin gene transcription (30 ). The cultures were then incubated with 1 µg/ml insulin or 0.5 mM 8-CPT-cAMP for 20 min, or left untreated as controls. Cytosolic (top) and nuclear (bottom) proteins were then prepared and analyzed by SDS-PAGE. The blots were sequentially probed with antibodies to PAK phosphorylated on Ser199/Thr423 (Cell Signaling Technology), total PAK (Transduction Labs, Lexington, KY), JNK phosphorylated on Ser78 (Cell Signaling Technology), and total JNK (Cell Signaling Technology). Probing against ERK (Santa Cruz Biotechnology) is shown as a loading control.

 
Jun N-terminal kinase (JNK) activation in response to insulin and cAMP was also examined because PAK was upstream of JNK in some signaling systems (32, 33). JNK activation by insulin or cAMP was determined in the presence or absence of LY294002 and U0126, and in untreated GH4 cells. Neither insulin nor cAMP increased JNK phosphorylation (Fig. 5Go). Interestingly, JNK phosphorylation was increased over control levels in LY294002 treated cultures and suggested that LY294002 could activate stress responses. This might explain the increase in basal prolactin-Luc expression sometimes seen in LY294002 treated culture because JNK phosphorylated and activated Elk1 (34). Finally, the JNK inhibitor SP600125 had no effect on basal or insulin-increased prolactin-Luc expression (data not shown). This made it unlikely that PAK effects were mediated through JNK.

PAK activation is affected by treadmilling inhibitors.
Activation of PAK by insulin should demonstrate the same sensitivity to inhibitors as insulin-increased prolactin gene expression if it mediates this response. Cytochalasin D, latrunculin B, jasplakinolide, and swinholide A decreased PAK activation by insulin (Fig. 6Go). Total levels of PAK and ERK1/2 were unaffected by treatment with the inhibitors.


Figure 6
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  		FIG. 6. PAK phosphorylation is inhibited by treadmilling blockers. GH4 cells were incubated with 2 µM cytochalasin D, 150 nM jasplakinolide, 2 µM latrunculin B, 40 nM swinholide A, or DMSO (vehicle) as control for 24 h. Cells were then pulsed with insulin for 20 min. Cytoplasmic (top) and nuclear (bottom) proteins were analyzed by SDS-PAGE and blotted to nitrocellulose. The blots were then sequentially probed with antibodies to PAK phosphorylated on Ser199/Thr423 (Cell Signaling Technology) and total PAK (Transduction Labs). Probing against ERK1/2 (Santa Cruz Biotechnology) is shown as a loading control.

 
Expression of TC10 N31, PAK LLKR, N-WASP {Delta}WH1, and Nck{alpha} SH2 and SH3 mutants inhibited insulin-increased prolactin-Luc activity. Therefore, these proteins should also block PAK activation in response to insulin. Figure 7Go shows that these mutant proteins significantly decreased activation of PAK by insulin in cytoplasmic extracts, whereas they completely blocked PAK activation in the nucleus (Fig. 7AGo). Cytoplasmic Akt activation by insulin was not significantly affected by expression of these plasmids, and only TC10 N31 and PAK LLKR significantly affected insulin activation of nuclear Akt (Fig. 7BGo). Thus, only PAK responded to these inhibitors in a way consistent with its mediating insulin-increased prolactin gene expression.


Figure 7
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  		FIG. 7. PAK phosphorylation in cells transfected with plasmids that inhibit insulin-increased prolactin gene expression. GH4 cells in 100-mm dishes were transfected with 10 µg CMV-TC10 N31, CMV-PAK LLKR, CMV-WASP {Delta}WH1, 5 µg CMV-Nck{alpha} SH2 mut/5 µg CMV-Nck{alpha} SH3 mut, or empty vector as a control. The cultures were treated with 1 µg/ml insulin for 30 min or left untreated as controls. Cytoplasmic and nuclear extracts were prepared as described previously, and the proteins were resolved by SDS-PAGE and blotted to nitrocellulose. The blots were probed with anti-phospho PAK 199/423 and anti-PAK (panel A), anti-phospho Akt Ser 473 and anti-Akt (panel B). The expression of tagged TC10 N31, N-WASP {Delta}WH1, PAK LLKR, and Nck{alpha} SH2 and SH3 mutants is shown in panel C. Anti-ERK1/2 was a loading control in all cases.

 
Insulin-increased prolactin-Luc expression is blocked by PAK knockdown in GH4 and T47D cells.
GH4 and T47D cells were treated with an siRNA designed to target rat PAK 1/3 because the insulin-responsive PAK migrates as either PAK1 or PAK3 on SDS-PAGE. This siRNA also had high homology to rat PAK2 and human PAK 1/3, and might reduce the levels of any/all of these proteins. Figure 8CGo confirms that PAK 1/3 levels are substantially reduced by this treatment in both cell types and that PAK2 is also reduced, although to a lesser extent. Luc expression was increased approximately 20-fold in control and scrambled siRNA treated GH4 cells, and approximately 10-fold in control and scrambled siRNA treated T47D cells (Fig. 8Go, A and B). This was significantly reduced more than 50% in cells treated with PAK1/3 siRNA (P < 0.01). The effect of cAMP was also significantly affected in T47D cells (P < 0.05), but not in GH4 cells.


Figure 8
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  		FIG. 8. Knockdown of PAK in GH4 and T47D cells using siRNA. GH4 and T47D cells were treated with 10 nM scrambled or PAK specific siRNA using Hiperfect (QIAGEN) for 24 h or untreated as control. Medium containing 10% charcoal-treated serum was then added, and the cells were retreated with siRNA. Three hours later, prolactin-Luc was transfected into GH4 cells using Genefector (Venn Nova) and T47D cells using Lipofectamine 2000 (Invitrogen). Insulin or forskolin was added, and the cells were incubated an additional 24 h. Luc activity was assessed as previously described (Fig. 1Go and Materials and Methods). A, GH4 cell Luc activity. B, T47D cell Luc activity. C, Western blot showing PAK expression in GH4 and T47D cells. PAK 1 and 3 are indistinguishable on Western blot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These data show that actin treadmilling is necessary for insulin-increased prolactin gene transcription and suggest that this could be due to treadmilling-dependent PAK activation. Treatments that disrupted the actin cytoskeleton of GH4 cells inhibited insulin-increased prolactin gene expression. Cytochalasin D, jasplakinolide, latrunculin B, and swinholide A treatment of GH4 cells disrupted the cortical actin layer and caused accumulation of actin adjacent to the plasma membrane (Fig. 2Go). Insulin-increased prolactin gene transcription was not observed under those conditions (Fig. 1Go). Activating forms of the Rho-related GTPases Rho-A (1), Cdc42 and TC10 (data not shown), altered the actin cytoskeleton of GH4 cells. This caused phenotypical changes in the GH4 cells and eliminated insulin-increased prolactin gene expression. These phenomena are relatively specific because higher levels of activated Rho/cytoskeletal rearrangement are required before cAMP or T3 signaling is affected (1).

Expression of dominant inhibitory mutants of Cdc42/TC10, which were incapable of mediating cytoskeletal rearrangement, blocked insulin-increased prolactin gene expression (Fig. 4AGo). Mutants of N-WASP, which is a downstream mediator of Cdc42/TC10, also blocked insulin-activated transcription (Fig. 4BGo). N-WASP nucleates the assembly of new actin filaments and was necessary for motility of Listeria in cells (9). N-WASP is likely to be involved in the movement of secretory granules in GH4 cells because it was shown that prolactin secretion was dependent on the rearrangement of cortical actin (36, 37, 38). This process would require remodeling of the cortical actin, which was both inhibitory to and required for secretion (37, 38).

The change from a stationary to a dynamic cytoskeleton could activate a kinase that then translocates to the nucleus to phosphorylate Elk1. Numerous kinases can activate Elk1. These experiments demonstrated that WT N-WASP and Nck{alpha} activate, whereas mutant forms of N-WASP and Nck{alpha} inhibit insulin-increased prolactin gene expression (Figs. 5Go and 6Go). This suggested that activation of N-WASP by Cdc42/TC10 initiates treadmilling and leads to activation of PAK, possibly through recruitment by Nck{alpha} to N-WASP. Activated PAK could then move into the nucleus to activate Elk1. This was supported by experiments that showed activation of prolactin-Luc expression by PAK WT and its inhibition by PAK mutants (Fig. 4DGo). PAK is activated by insulin in GH4 cells, and PAK activation was appropriately sensitive to LY294002 and not to U0126 (Fig. 5Go). Furthermore, activated PAK is found in the nucleus (Fig. 5Go), and PAK phosphorylated the transcription factor Forkhead related, which is involved in insulin regulation of many promoters (39). PAK is also appropriately sensitive to inhibition by treadmilling inhibitors (Fig. 6Go) and mutant proteins (Fig. 7Go). It could fulfill this role, or it could activate another kinase that phosphorylated Elk1.

It is interesting that most of the signaling enzymes that were once thought to be obligatorily cytoplasmic have now been found to act in the nucleus. Lim kinase mediated cyclin D1 expression (40). Rock2 phosphorylated p300 and activated its acetyltransferase activity (41). PI 3-kinase and the counter-regulatory enzyme phosphatase and Tensin homolog have both had an important regulatory function in the nucleus (42). Even N-WASP had effects that were due to its nucleation of actin filaments in the nucleus (43). These studies make it clear that cytoplasmic/nuclear signaling loops are the norm and that treadmilling-dependent kinase activation should be expected to have nuclear consequences.

These studies are reminiscent of studies of insulin-stimulated glucose transporter translocation and glucose-dependent insulin secretion. Those processes were also dependent on cytoskeletal remodeling. Glucose transporter 4 translocation in response to insulin, but not platelet-derived growth factor, was treadmilling dependent (44), and shown to be N-WASP dependent in skeletal muscle in vivo (45). Insulin secretion is also dependent on cytoskeletal rearrangement (46, 47, 48). Glucose-stimulated insulin secretion was inhibited in a cell line with excess F-actin, and this could be reversed using latrunculin B to depolymerize F-actin. These studies suggest that cytoskeletal rearrangement may be a common theme in insulin signaling.

Treadmilling might also affect prolactin gene transcription by increasing the level of G actin. Actin and actin-like molecules are components of several chromatin-remodeling complexes, and excess G actin could sequester essential components of one of these complexes and squelch their activity. One such complex was the NuA4/Tip60 complex, which was shown to contain TRRAP, the enhancer of polycomb protein, actin-like protein BAF53a, actin, Tip60, the SNF2-related helicase p400 and Tip49a and b (49). The SWI/SNF related BAF complex also contains both actin and the actin-related protein Baf53. The actin in this complex is so tightly bound that it is not possible to dissociate the actin without denaturing the complex (16, 50, 51). In addition, this complex was shown to bind to both chromatin and actin filaments in a phosphoinositide-dependent manner. Activation of these complexes by actin has not been shown, but this could result from abnormal localization of exogenously expressed actin. Follow-up of this hypothesis requires the identification of chromatin remodeling complexes involved in prolactin promoter activation.

These results are also consistent with a model in which the breakdown and resynthesis of actin filaments liberate a factor to the nucleus that activates specifically prolactin promoter expression. This factor could be a transcription factor or a transcriptional coactivator. In this model the amounts of F and G actin in the cell would remain relatively constant, but the filaments would be in a dynamic rather than a static condition. This was extensively studied using the expression of SRF responsive promoters (12, 13). MAL, a coactivator for SRF, is librated from the actin cytoskeleton by treadmilling and translocates to the nucleus, where it activates a subset of promoters. Elk1was identified as the Ets-related transcription factor that mediated the effect of insulin on the prolactin promoter (30), and it is also a ternary complex factor involved in c-fos promoter activation. The data demonstrating the treadmilling dependent sensitivity of the c-fos promoter clearly implicated SRF and not ternary complex factors Elk1 and Sap1 (12), but it does not exclude the possibility of a different Elk1/prolactin specific factor that is treadmilling dependent.

The contrasting sensitivities of the prolactin and GH promoters to perturbations of actin treadmilling are similar to their divergent response to Ca2+ mobilization (28). Prolactin is significantly more sensitive than GH to changes in cellular Ca2+. Gelsolin is a Ca2+-dependent F-actin severing protein that might provide a link between these data and ours. Interestingly, the c-Fos promoter was also Ca2+ sensitive (29), and the activity of SRF that activates the c-fos promoter is dependent on actin treadmilling (12).

The activation of Cdc42/TC10 by insulin is an important issue that remains unresolved by these and other insulin-signaling studies. A pathway using APS, Cbl, CAP, Crk, and C3G had been proposed (5), but subsequent studies suggested that this was not likely correct (35). Our previous studies suggested that Ras activation might play a role in insulin-increased prolactin gene transcription that was not mediated through Erk (3). It is possible that Ras recruits an exchange factor for Cdc421/TC10. Alternately, an adapter such as Grb2 or Nck might recruit a Cdc42/TC10 exchange factor to a phosphorylated IRS.

Integrin and insulin produced an additive activation of prolactin gene transcription (1). The effect of integrin was mediated through receptor-like protein tyrosine phosphatase{alpha}, Src and Rho. Activation of Rho caused alteration of the actin cytoskeleton and activated prolactin promoter expression. It is possible that PAK is activated by integrin-mediated cytoskeletal alteration and that PAK is part of a common final pathway for activation of prolactin gene expression. Alternately, integrin may activate a different kinase, e.g. integrin linked kinase that has additive effects with PAK. Further studies will be needed to demonstrate clearly the dependence of insulin-increased gene expression on cytoskeletal mobilized PAK and any interrelation with the integrin signaling pathway.


   Acknowledgments
 
I thank M. Rush (New York University School of Medicine, New York, NY) for plasmids used in these studies.


   Footnotes
 
The Research Computing Resource at New York University School of Medicine, which is supported through Grant BIR-9318128 from the National Science Foundation, was used for database support.

Disclosure Summary: The author has nothing to declare.

First Published Online September 20, 2007

Abbreviations: Akt, Protein kinase B; CMV, cytomegalovirus; GSK, glycogen synthase kinase; IRS, insulin-receptor substrate; JNK, jun N-terminal kinase; Luc, luciferase; mTOR, mammalian target of rapamycin; N-WASP, neuronal Wiskott-Aldrich syndrome protein; PAK, p21 associated kinase; PI, phosphatidylinositol; siRNA, small interfering RNA; SRF, serum response factor; WT, wild type.

Received January 29, 2007.

Accepted for publication September 4, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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