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CELLULAR AND MOLECULAR
The Cell Signalling Laboratory, Hawthorn Building, Leicester School of Pharmacy, De Montfort University, Leicester, England
Received February 7, 2007; accepted March 15, 2007.
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Abstract |
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). Currently, however, only limited proliferation and retention of function of primary human or rat hepatocytes in culture has been achieved on stimulation with growth factors (Loyer et al., 1996; Hino et al., 1999; Runge et al., 1999, 2000a,b; Katsura et al., 2002; Paine and Andreakos, 2004; Serandour et al., 2005; Yamasaki et al., 2006). The extent of this proliferation is insufficient to produce large enough populations of fully functional hepatocytes to be used therapeutically. An increased understanding of how growth factor receptors on the cell surface signal toward the nucleus to control cell cycle progression in hepatocytes is therefore of obvious importance. Here, we have studied stimulation with insulin and EGF under conditions that maximize rapid entry of rat hepatocytes into the cell cycle. Hepatocytes are highly differentiated, multifunctional parenchymal cells. The retention of hepatocyte characteristics is a requirement of the proliferated cells. One unique and highly regulated function of hepatocytes is to store glucose in the form of glycogen and to release it into the bloodstream when required. This release is controlled by glycogen phosphorylase, the activation of which is under dual control by Ca2+ and cyclic AMP signaling pathways. Here we have examined proliferating cultures of rat hepatocytes for evidence that these crucial signaling pathways remain fully functional and that coupling to the activation of glycogen phosphorylase remains intact.
It has been shown that stimulation of hepatocytes with insulin and EGF leads to activation of Akt through PI3K (Band et al., 1999; Roberts et al., 2000; Coutant et al., 2002; Ribaux et al., 2002; Thoresen et al., 2003; Schulze-Bergkamen et al., 2004). Activation of this signaling pathway is known to play a role in restoration of liver mass after partial hepatectomy (Haga et al., 2005). The mTOR pathway is also downstream of PI3K and can be activated either independently of Akt or directly by insulin-activated Akt (Nave et al., 1999). Inhibition of mTOR with rapamycin attenuates hepatocyte responses to insulin and EGF (Francavilla et al., 1992; Band et al., 1999; Coutant et al., 2002; Kim et al., 2006). However, it has also been reported that attenuation of the Akt component of the PI3K cascade in hepatocytes, by expression of dominant-negative Akt, does not inhibit EGF-stimulated DNA synthesis (Coutant et al., 2002). This finding has led to the suggestion that mTOR, rather than Akt, regulates the proliferative responses in hepatocytes. Here we have used the inhibitor of Akt, A-443654 (Luo et al., 2005; Shi et al., 2005), to investigate directly for the first time the role of Akt in proliferative responses to insulin and EGF in primary hepatocytes. In addition, we have examined the consequences of adenovirus-mediated expression of both dominant-negative, and constitutively active Akt.
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Materials and Methods |
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[3H]Thymidine Incorporation into DNA and MTT Assay. Cells were cultured under the conditions indicated in 24-well plates, and for the final 4 h with 1 µl/ml methyl-[3H]thymidine (activity = 37 MBq/ml: GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). Cells were preincubated with inhibitors [LY294002 (Calbiochem, San Diego, CA), rapamycin (Sigma-Aldrich, St. Louis, MO), and A-443654 (kind gift of Dr. Yan Luo, Abbott Laboratories (Abbott Park, IL)] for 15 to 30 min before addition of either insulin or EGF. The concentration of A-443654 used here (3 µM) was chosen as the minimal concentration required to inhibit EGF-stimulated [3H]thymidine incorporation into DNA (preliminary experiments, data not shown). Control experiments indicated that up to 0.5% dimethyl sulfoxide had no measurable effect; it did not exceed 0.2% in the experiments reported here. The MTT cell assay for volume of viable cells was carried out according to the manufacturer's (Promega, Madison, WI) instructions in parallel with [3H]thymidine experiments.
Immunocytochemistry. Cells were cultured on collagen-coated Thermanox coverslips (Nalge Nunc International (Rochester, NY). After BrdU incorporation (15 µMfor the last 4 h of culture), cells were fixed with formalin, permeabilized with methanol, and incubated with rabbit anti-human albumin (Sigma-Aldrich) and monoclonal anti-BrdU antibodies (Calbiochem). After incubation with fluorochrome-conjugated secondary antibodies (Cy5-conjugated donkey anti-rabbit and Cy3-conjugated goat anti-mouse from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA), cells were visualized with a Leica SM2 confocal imaging system (Leica Microsystems, Heidelburg GmbH, Germany). Controls with no primary antibody showed essentially no fluorescence above background autofluorescence.
Western Blots. After the culture times indicated, cells were lysed (20 mM Tris-HCl, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 1 mM 
-mercaptoethanol, 20 µg/ml aprotinin, and 5 µg/ml leupeptin, pH 7.6), and proteins were separated on 10% polyacrylamide gels. Blots were probed with anti-phospho Akt (Ser473) or anti-phospho GSK-3, both from Cell Signaling Technology Inc. (Danvers, MA, and visualized using ECL+ Plus (Amersham Biosciences UK, Ltd.).
Measurement of [Ca2+]c. Cells cultured on collagen-coated coverslips were loaded with fura 2/acetoxymethyl ester and imaged for Ca2+c in a continuous perfusion chamber as described previously (Dixon et al., 2005). Agonist stimulation was for 30 s. Stock solutions (10 mM) of ATP and UTP were incubated with a regenerating system comprising 10 mM creatine phosphate and 20 U/ml creatine phosphokinase. UDP was treated with 80 U/ml hexokinase and 110 mM glucose.
Glycogen Phosphorylase and Total [3H]InsPx. Cells were cultured in six-well plates and assayed for glycogen phosphorylase activity as described previously (Dixon et al., 2005). For total [3H]In-sPx, cells were cultured in 24-well plates for 4 h and then labeled for 44 h with myo-[2-3H]inositol in serum-free WME (0.185 Mbq/ml, 0.5 ml/well). Twenty-minute stimulations in the presence of 10 mM LiCl were performed without change of medium. Total [3H]InsPx was extracted as described (Dixon et al., 2005).
Adenoviral Expression of Dominant-Negative Akt and myrAkt. Cells were cultured for 4 h as described. Medium was replaced with serum-free WME containing adenovirus encoding kinase-dead dominant negative (dn) Akt or constitutively active myrAkt. Expression levels resulting from different dilutions of adenovirus were monitored by Western blotting for panAkt to establish a consistent level of Akt expression across the experiments undertaken. These panAkt Western blots showed that expression was apparent at 24 h, maximal at 48 h, and declined thereafter. The adenovirus remained in the cultures, EGF was added where appropriate 20 h later, and [3H]thymidine and MTT assays were completed after a further 24 h. The adenoviral constructs were a kind gift of Dr. R. A. Rippe, University of North Carolina, Chapel Hill, NC.
Data Analysis. Data pooled from separate experiments were collected from different cell preparations and analyzed by one-way ANOVA with Bonferroni's or Dunnett's post-test as indicated, using GraphPad Prism (GraphPad Software Inc., San Diego, CA).
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Results |
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50% at 96 h as illustrated in Fig. 1B. In comparison, cultures stimulated with insulin and EGF displayed an increased protein content over the first 48 h to 1.52 ± 0.19-fold higher than controls (mean ± S.E., n = 3 separate experiments). Passage of cells into S phase was also assessed through BrdU incorporation; cells were coimmunostained for albumin to confirm their identity as hepatocytes (Fig. 1C). After 4 h in culture, essentially all cells stained positively for albumin and revealed no incorporation of BrdU (left panel). After 48 h in culture and addition of EGF at 24 h, there was both an increase in the area occupied per cell and in cell number (typically 1.5-fold by 48 h); hepatocytes staining positively for BrdU could be clearly seen (middle panel). Hepatocytes were occasionally captured with BrdU-stained chromosomes in anaphase (right panel). These experiments established that the conditions used gave reliable stimulation of hepatocytes into S phase.
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Receptor-Regulated Events in Insulin/EGF-Treated Hepatocytes. To determine whether the insulin/EGF-treated cells retain characteristics of hepatocytes, we studied the signaling pathways leading to glycogen phosphorylase activation. In previous studies we have characterized increases in [3H]InsPx and [Ca2+]c in rat hepatocytes in response to extracellular nucleotides acting through P2Y1 and P2Y2 receptors (Dixon, 2000; Dixon et al., 2004, 2005). Here we have shown that accumulation of [3H]InsPx in response to 2-methylthio-ADP (acting through the P2Y1 receptor) and ATP and UTP (P2Y2) are not significantly different in control and insulin/EGF-treated cells (data not shown). In addition individual cells from these two populations produce indistinguishable rises in [Ca2+]c, in terms of both size and concentration dependence, when stimulated by these nucleotides (data not shown). Consistent with these findings, no differences were observed in the activation of glycogen phosphorylase in response to the nucleotides ATP, UTP, 2-methylthio-ADP and UDP in populations of control and proliferating cells (Fig. 2). Activation of glycogen phosphorylase by glucagon, which is mediated by increased cyclic AMP levels, was also unaltered as seen in Fig. 2. For both glycogen phosphorylase and [3H]InsPx assays, the absolute values per well were higher in the insulin/EGF-treated cultures, reflecting the greater cell content per well (not shown).
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; Coutant et al., 2002), both drugs blocked stimulation of DNA synthesis by EGF (data not shown). For LY294002 this inhibition showed a dose dependence similar to that for inhibition of EGF-stimulated Akt phosphorylation; [3H]thymidine incorporation in response to 20 ng/ml EGF was reduced to 23.9 ± 5.2% by 5 µM LY294002 and to 3.4 ± 1.7% by 20 µM LY294002 (mean ± S.E.M., n = 3 separate experiments). Rapamycin (3 nM) completely abolished the response to 20 ng/ml EGF (not shown). The inclusion of the Akt inhibitor, A-443654 (3 µM), during the 4-h incubation with insulin, led to a reduction in [3H]thymidine incorporation at 48 h in both control and insulin-stimulated cells (Fig. 4C). To determine whether this reduction was due to cell death, we also measured protein concentrations of cultures and used the MTT assay to assess numbers of viable cells. When measured after 48 h in culture, protein levels (not shown) and cell viability (Fig. 4D) were reduced by inclusion of A-443654 in the initial 4-h culture period. With inhibitor present for 48 h, there was a major loss of both protein and MTT activity, and the cells became rounded and apoptotic (not shown).
The effect of A-443654 on the EGF-mediated stimulation of DNA synthesis was investigated. A-443654 (3 µM) was added 30 min before the addition of EGF after 24 h in culture. Cells were then exposed to EGF for 24 h and to A-443654 for varying periods, after which inhibitor was removed and EGF was replaced. EGF significantly stimulated the [3H]thymidine incorporation into DNA 20 to 24 h later. When EGF was added in the presence of A-443654, there was no significant stimulation, even when the A-443654 was only present for the 1st h of the 24-h period of EGF stimulation (Fig. 5A). Of importance, the 1-h exposure to A-443654 did not lead to significant changes in protein concentration, cell viability (as assessed by MTT assay), or appearance of cells after 24 h (not shown). To investigate whether A-443654 inhibits Akt in these cells, Western blots for phospho-GSK-3, a substrate of the Akt kinase, were performed; Figure 5B shows a representative blot, and Fig. 5C shows densitometric data pooled from four experiments. Both EGF and insulin stimulated the phosphorylation of GSK-3; this was blocked by 3 µM A-443654.
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Discussion |
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In the initial work described above, we have defined culture conditions that stimulate cell cycle progression. The resulting population of cells was assessed for the retention of hepatocyte characteristics through the study of glycogen phosphorylase activation. This key enzyme in glycogenolysis is activated by glucagon mediated by increases in cyclic AMP levels and by extracellular nucleotides, acting via members of the P2Y family of receptors to increase [Ca2+]c (Okajima et al., 1987; Keppens et al., 1992, 1993; Dixon, 2000; Carini et al., 2006). The complex pattern of second messenger increases and glycogen phosphorylase activation effected by glucagon and a range of nucleotides acting through multiple P2Y receptors was similar in cells from control cultures and those progressing through the cell cycle.
We then set out to investigate the signaling mechanisms from insulin and EGF receptors that bring about the proliferative response in these hepatocyte cultures. An early report (Band et al., 1999) suggested that the proliferative response to EGF is independent of the activation of extracellular signal-related kinase and that activation of PI3K/Akt/mTOR is sufficient. However, later evidence (Coutant et al., 2002; Thoresen et al., 2003) indicated that both extracellular signal-related kinase and PI3K pathways are required downstream of the EGF receptor. Furthermore, on the basis of a failure of dominant negative Akt to abrogate EGF-stimulated DNA synthesis, it was concluded that Akt was not directly involved in the cell division stimulated by EGF (Coutant et al., 2002). This conclusion supported the notion that the PI3K-dependent response is mediated by the mTOR branch of the pathway and not by Akt. With the recent development of a chemical inhibitor of Akt (A-433654) (Luo et al., 2005; Shi et al., 2005), we decided to further investigate the role of elements of the PI3K/mTOR/Akt pathway in the responses to insulin and EGF, which establish the proliferating cultures described above.
In agreement with earlier work (Coutant et al., 2002; Ribaux et al., 2002; Kim et al., 2006), we have demonstrated that Akt is phosphorylated in response to EGF and insulin; this phosphorylation was transient in response to EGF (peak at 5–10 min) and sustained (over 24 h) in response to insulin. Interestingly, the inclusion of insulin for only the first 4 h of culture resulted in enhanced phosphorylation of Akt 24 h later when EGF was added. This sustained activation of the PI3K pathway may contribute to the lasting influence of insulin, long after its removal from culture. The insulin and EGF-stimulated phosphorylation of Akt and GSK-3 was eliminated by the inhibition of the PI3K pathway by LY294002. We then showed that LY294002 inhibited insulin-stimulated DNA synthesis, confirming the requirement for the PI3K cascade in progression through the cell cycle. Similarly the significance of mTOR in these cultures is clearly demonstrated by the effects of the inhibitor, rapamycin; [3H]thymidine incorporation was reduced in both control and insulin-treated cultures. However, the clear residual response to insulin indicates that although mTOR plays a role in proliferation of hepatocytes, stimulation by insulin is, in part, independent of mTOR.
A-443654 inhibited [3H]thymidine incorporation similarly in both control and insulin-enhanced cultures, at a concentration that we have shown to eliminate agonist-stimulated phosphorylation of the Akt substrate, GSK-3. Down-regulation of Akt has been associated with apoptosis in hepatocytes (Schulze-Bergkamen et al., 2004). It was therefore important to determine whether the attenuation of [3H]thymidine incorporation could be accounted for by cell death. The measurements of the protein content of cultures and cell viability through the MTT assay were consistent with an increased loss of cells when A-443654 was present during the first 4 h of culture. Overall the initial inhibition of DNA synthesis by A-443654, followed by an increasing loss of cells over 48 h, are consistent with a requirement for Akt for both cell cycle progression, and to restrain apoptosis.
The experiments investigating the effect of Akt inhibition on EGF-stimulated cell cycle progression had a clear outcome. When cells were stimulated with EGF for 24 h, A-443654 needed to be present only during the 1st h to have a maximal effect, eliminating EGF-stimulated cell cycle progression. This is consistent with the transient stimulation of Akt by EGF seen here in Western blots. Under these conditions, with A-443654 present for only 1 h, there was no subsequent loss of viable cells over the next 24 h, indicating that the Akt inhibitor did not enhance apoptosis. These results provide a compelling case for an obligatory role for Akt in the stimulation by EGF of cell cycle progression in hepatocytes, independent of the effect that inhibition of this kinase may have on apoptosis. Interestingly, though, we have found that the maximal stimulation of DNA synthesis by the addition of EGF 20 h after plating cells with insulin, requires that EGF be present for >8 h (Y. Luo and M. R. Boarder, unpublished data). The most likely explanation is that persistent signaling from the EGF receptor through other pathways, such as sustained extracellular signal-related kinase activation (Thoresen et al., 2003), is required in addition to the transient Akt activation.
The adenoviral-mediated expression studies presented here are consistent with a central role of Akt in EGF-stimulated cell cycle progression. Three notable observations were made. First, the expression of myrAkt led to enhanced [3H]thymidine incorporation, second, the expression of dnAkt reduced the [3H]thymidine incorporation stimulated by EGF, and third, the expression of myrAkt and stimulation with EGF were not additive. Furthermore, it is characteristic of our experiments with EGF stimulation that a substantial [3H]thymidine response was seen with little or no increase in the MTT response, consistent with the cells passing through S phase at this time but not completing proliferation. In contrast, the expression of constitutively active Akt increased both the [3H]thymidine response and the number of viable cells as assessed by the MTT assay. These observations are consistent with cells having completed replication at this time point, 44 h from the start of transfection. Through Western blots we have shown that expression of heterologous Akt protein is apparent at 24 h and increased after 48 h. Our heterologous expression studies suggest that sustained elevation of Akt alone is sufficient to elicit complete transit through the cell cycle. This contrasts with the conclusions for stimulation by EGF described above, for which a transient stimulation of Akt is necessary, but not sufficient, to stimulate cell cycle progression. A reduction in apoptosis as a result of a sustained elevation in Akt activity may also contribute to the enhanced MTT reading with myrAkt treatment.
In conclusion, we have demonstrated that the Akt element of the PI3K cascade is necessary for EGF-stimulated cell cycle progression. The sustained elevation of Akt through adenovirus-mediated Akt expression generates a hyperproliferative window in hepatocyte cultures, increasing the possibility of achieving the goal of producing expanding populations of fully functional hepatocytes for future cell therapy procedures. Further studies will characterize the hepatocyte population after adenovirus-mediated expression of myrAkt.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: EGF, epidermal growth factor; PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; A-443654, ·; WME, William's medium E; FCS, fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; BrdU, bromodeoxyuridine; [Ca2+]c, cytosolic free calcium concentration; InsPx, inositol (poly)phosphates; dn, dominant-negative; GSK-3, glycogen synthase kinase 3; ANOVA, analysis of variance; A-443654, (S)-1-(1H-indol-3-yl-methyl)-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine.
Address correspondence to: Dr. Michael R. Boarder, The Cell Signaling Laboratory, The Hawthorn Building, Leicester School of Pharmacy, De Montfort University, Leicester LE1 9BH, UK. E-mail: mboarder{at}dmu.ac.uk
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) and insulin/EGF-stimulated cultures (
). C, simultaneous staining for albumin and BrdU in rat hepatocytes after 4 h of culture with 10 µg/ml insulin in the presence of BrdU (left panel) or 48 h culture with insulin and EGF as above (middle and right panels).
