Introduction

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Liver regeneration in response to partial hepatectomy or chemical liver injury is a physiological growth response observed in intact animals (Sandnes et al., 1986; Michalopoulos, 1990). During liver regeneration, quiescent hepatocytes undergo one or two rounds of replication and then return to a nonproliferative state. Growth factors regulate this process by providing both stimulatory and inhibitory signals for cell proliferation. A variety of growth factors, including EGF and HGF, have potent mitogenic effects on hepatocytes and stimulate normal liver growth and liver regeneration (Nakamura et al., 1983b). HGF is a potent mitogen first purified from rat platelet and human and rabbit plasma (Nakamura et al., 1986, 1987, 1989). The response of adult rat hepatocytes to HGF and other growth factors has been studied extensively with respect to DNA synthesis and proliferation in vitro (Richman et al., 1976; McGowan et al., 1981; Nakamura et al., 1983a; Marker et al., 1992). However, such experiments were performed during the relatively late phases of culture (i.e., 24- 48 h).

We have reported previously that EGF and insulin alone can rapidly stimulate hepatocyte DNA synthesis and proliferation during short-term (i.e., approximately 4 h) cultures (Kimura and Ogihara, 1997a; Kimura and Ogihara, in press, 1997b). Depending on the growth factor, hepatocyte proliferation is dependent on the plating density. For example, hepatocyte DNA synthesis and proliferation induced by EGF is strictly dependent on the initial plating density, whereas that induced by insulin does not depend exclusively on initial plating density. Furthermore, hepatocyte proliferation appears to be potentiated by beta adrenergic agonists and other cAMP-elevating agents.

Recently, the signal transduction pathway activated in response to HGF in hepatocytes has become understood more clearly (Marker et al., 1992; Gines et al., 1995). HGF initiates its proliferative effects through the activation of tyrosine kinase-linked receptors and can induce replication in adult rat hepatocytes (Osada et al., 1992). However, the precise mechanism by which HGF acts remains unclear. Thus, the present study investigated the possibility that HGF alone also participates in the intracellular events involved in rapid proliferation of adult rat hepatocytes. In addition, we examined the effects of alpha and beta adrenergic agonists on HGF-induced DNA synthesis in adult rat hepatocytes to clarify the relationship between HGF action and the adrenergic responses. Finally, we investigated pharmacologically the cell signaling systems involved in the HGF responsiveness in primary cultures of adult rat hepatocytes.

	Materials and Methods

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Hepatocyte isolation and culture. Male Wistar rats (weight 200-250 g) were obtained from Saitama Experimental Co. (Saitama, Japan). The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (45 mg/kg). A two-step in situ collagenase perfusion was performed to facilitate disaggregation of the adult rat liver as described previously (Seglen, 1975; Ogihara, 1995). The liver was first washed via the portal vein with Ca⁺⁺-free Hanks-10 mM HEPES buffer (pH 7.4) at 37°C and a flow rate of 30 ml/min for 10 min. The second step was performed with use of the same buffer containing 0.025% collagenase and 0.075% CaCl₂ at a flow rate of 30 ml/min for 10 min. The cells were dispersed in Ca⁺⁺-free Hanks' solution. The cells were then washed three times by slow centrifugation (120 × g) for 1 min to remove cell debris, damaged cells and nonparenchymal cells. The viability of hepatocytes was monitored by trypan blue dye exclusion. On average, more than 94% of the cells remained intact. Unless otherwise indicated, isolated hepatocytes were plated onto plastic culture dishes (Sumitomo Bakelite Co., Tokyo, Japan) at a density of 3.3 × 10⁴ cells/cm² in Williams' medium E containing 5% bovine calf serum, 10¹⁰ M dexamethasone for 3 h in 5% CO₂ in air. The medium was then changed, and the cells were cultured in serum- and dexamethasone-free Williams' medium E containing various concentrations of HGF with or without beta adrenergic agonists, cAMP-elevating agents, an alpha-1 adrenergic agonist and/or specific inhibitors of signal transducers.

Measurement of DNA synthesis. Hepatocyte DNA synthesis was assessed by measuring [³H]thymidine incorporation into acid-precipitable materials (Morley and Kingdon, 1972). After an initial attachment period of 3 h, the hepatocytes were washed twice with serum-free Williams' medium E and cultured in a medium containing 5 ng/ml HGF for a further 4 h and 21 h. The cells were pulsed at 2 h and 19 h post-HGF stimulation for 2 h with [³H]thymidine (1.0 µCi/well). Incorporation into DNA was determined as described previously (Kimura and Ogihara, 1997a). The hepatocyte protein content was measured by a modified Lowry procedure with bovine serum albumin as a standard (Lee and Paxman, 1972).

Counting nuclei. The number of nuclei was counted instead of the cell number according to the previously described procedure of Nakamura et al. (1983a) with minor modifications. The cultured hepatocytes were washed twice with 2 ml of Dulbecco's phosphate-buffered saline (pH 7.4). Then, the cells were lysed by incubation with 0.25 ml of 0.1 M citric acid containing 0.1% Triton X-100 for 30 min at 37°C. An equal volume of the nucleus suspension was mixed with 0.3% trypan blue in Dulbecco's phosphate-buffered saline and the number of nuclei was counted in a hemocytometer. This procedure was performed because the hepatocytes firmly attached to the collagen-coated plates and were not dispersed by EDTA-trypsin treatment.

Materials. The following reagents were obtained from Sigma Chemical Co. (St. Louis, MO): HGF (human recombinant), forskolin, db-cAMP, genistein, forskolin, aphidicolin, metaproterenol hemisulfate, butoxamine hydrochloride, metoprolol tartrate, dobutamine hydrochloride, phenylephrine hydrochloride, D-sphingosine, ionomycin calcium salt, UK14304, glucagon (porcine), wortmannin, rapamycin and dexamethasone. H-892HCl and U-73122 were obtained from BIOMOL, Research Laboratories Inc. (Plymouth Meeting, PA). PMA was purchased from Research Biochemicals International (Natick, MA). Williams' medium E and newborn calf serum were purchased from Flow Laboratories (Irvine, Scotland). Collagenase (type II) was obtained from Worthington Biochemical Co. (Freehold, NJ). [methyl-³H]Thymidine (20 Ci/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). All reagents were of analytical grade.

Statistical analysis. Values are expressed as mean ± S.E.M. Data were analyzed by the unpaired Student's t test. P values less than 0.05 were regarded as statistically significant.

	Results

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Time course associated with stimulation of hepatocyte DNA synthesis and proliferation induced by HGF with or without metaproterenol. Isolated adult rat hepatocytes were treated with HGF (5 ng/ml) with or without metaproterenol (10⁷ M) at various points during the culture period, and DNA synthesis was measured by [³H]thymidine incorporation at a low cell density (3.3 × 10⁴ cells/cm²). DNA synthesis was induced in hepatocytes after only 2 h and reached a maximum 3 h after adding HGF (5 ng/ml). However, DNA synthesis became markedly reduced at 21 h (fig. 1). Hepatocyte DNA synthesis induced by HGF was potentiated in the presence of a beta-2 adrenergic agonist, metaproterenol (10⁷ M) and a nonspecific beta adrenergic agonist, isoproterenol (10⁷ M, not shown), during the early phase of culture. The HGF (5 ng/ml)-induced increase in the number of nuclei (proliferation) began approximately 3.5 h after the addition of HGF and gradually increased for a further 17 h. Proliferation was potentiated by metaproterenol treatment. Therefore, the detected increase in the number of nuclei could be caused by an increase in the [³H]thymidine incorporation after HGF treatment.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Time course of stimulation of hepatocyte DNA synthesis and proliferation by HGF with or without metaproterenol (Meta). Hepatocytes (3.3 × 104 cells/cm2) were cultured with or without metaproterenol (107 M) in the presence of HGF (5 ng/ml) for various lengths of time. Data are expressed as the mean ± S.E. of three separate experiments. *P < .05, **P < .01 compared with controls (values just before HGF addition).

Time course associated with stimulation of hepatocyte DNA synthesis and proliferation induced by HGF with or without phenylephrine. Isolated adult rat hepatocytes were treated with HGF (5 ng/ml) with or without phenylephrine (10⁶ M) at various points during the culture period, and DNA synthesis was measured by [³H]thymidine incorporation at a low cell density (3.3 × 10⁴ cells/cm²). DNA synthesis was induced in hepatocytes after only 2.5 h and reached a maximum 3 to 4 h after the addition of HGF (5 ng/ml). Proliferation became markedly reduced by 21 h (fig. 2). Hepatocyte DNA synthesis induced by HGF was potentiated in the presence of an alpha-1 adrenergic agonist, phenylephrine (10⁶ M) during early phase of culture. The HGF (5 ng/ml)-induced increase in the number of nuclei (proliferation) began approximately 3.5 h after the addition of HGF and gradually increased for a further 17 h. Proliferation was potentiated by phenylephrine treatment.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Time course of stimulation of hepatocyte DNA synthesis and proliferation by HGF with or without phenylephrine (Pheny). Hepatocytes (3.3 × 104 cells/cm2) were cultured with or without phenylephrine (106 M) in the presence of HGF (5 ng/ml) for various lengths of time. Data are expressed as mean ± S.E. of three separate experiments. *P < .05, **P < .01 compared with controls (values just before HGF addition).

Effect of dexamethasone pretreatment on HGF-induced hepatocyte DNA synthesis and proliferation during early and late phases of culture. To investigate the mechanism by which HGF rapidly stimulates hepatocyte DNA synthesis and proliferation, we examined the effects of dexamethasone pretreatment (3 h after plating) on HGF-stimulated hepatocyte DNA synthesis and proliferation during the early and late phases of culture. Figure 3A shows that HGF-induced hepatocyte DNA synthesis and proliferation were greatly impaired at the stage of 4-h culture when relatively large doses of dexamethasone (10⁸ and 10⁷ M) were added during the 3-h attachment period. The inhibitory effects of dexamethasone on HGF-stimulated hepatocyte DNA synthesis and proliferation (IC₅₀ 2.7 ± 0.3 nM) were partially restored to the control level after culture with HGF for 21 h (fig. 3B).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of dexamethasone pretreatment on HGF-induced hepatocyte DNA synthesis and proliferation during early and late phases of culture. Hepatocytes (3.3 × 104 cells/cm2) were cultured with or without various concentrations of dexamethasone (1010-107 M) in the absence of HGF for 3 h. The medium was then changed, and the cells were cultured in serum- and dexamethsone-free Williams' medium E containing 5 ng/ml HGF. Data are expressed as mean ± S.E. of three separate experiments. *P < .05, **P < .01 compared with respective controls.

Dose-dependent effect of HGF on hepatocyte DNA synthesis and proliferation. Dose-response effects of HGF on hepatocyte DNA synthesis and proliferation in the low-density culture (3.3 × 10⁴ cells/cm²) for 4 h were examined. As shown in figure 4, the effect of HGF on hepatocyte DNA synthesis was dose-dependent. Peak stimulation of hepatocyte DNA synthesis was seen at the dose of 3 ng/ml and showed an EC₅₀ of 0.95 ± 0.09 ng/ml. The number of nuclei increased dose-dependently by approximately 1.3-fold with HGF administration. The maximal effect of stimulation occurred at approximately 5 ng/ml and showed an EC₅₀ of 0.93 ± 0.01 ng/ml (fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Dose-dependent effect of HGF on hepatocyte DNA synthesis and proliferation. Hepatocytes (3.3 × 104 cells/cm2) were cultured with various concentrations of HGF for 4 h. Data are expressed as mean ± S.E. of three separate experiments.

Influence of cell density on HGF-stimulated hepatocyte DNA synthesis and proliferation with or without metaproterenol and phenylephrine. To study whether or not the proliferative effect of HGF is affected by the initial plating density, we investigated the density dependence of hepatocyte DNA synthesis and proliferation induced by 5 ng/ml HGF with or without metaproterenol or phenylephrine. Figure 5 shows that initial plating density appears to influence an important step involved in hepatocyte DNA synthesis. Hepatocyte DNA synthesis was induced by HGF at low densities, but became markedly reduced at a high cell density that approaches confluence, both in the presence and absence of metaproterenol or phenylephrine. As shown in figure 6, the HGF (5 ng/ml)-induced increase in the number of nuclei reached a plateau at a cell density of 3.3 × 10⁴ cells/cm². The HGF-induced increase was observed both during the early and late phases of culture (not shown). However, the effects of HGF treatment were reduced or absent at a high cell density, regardless of the presence or absence of metaproterenol or phenylephrine. Hepatocyte DNA synthesis and proliferation in hepatocytes cultured without or with dexamethasone (10¹⁰ M) for 21 h did not appear to be affected, regardless of cell density.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Influence of cell density on metaproterenol (Meta) (107 M)- or phenylephrine (Pheny) (106 M)-stimulated hepatocyte DNA synthesis in presence of HGF. Hepatocytes were cultured with dexamethasone (1010 M) or HGF (5 ng/ml) at various plating densities for 4 h. Data are expressed mean ± S.E. of three separate experiments. *P < .05, **P < .01, ***P < .001 compared with the respective controls.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Influence of cell density on the metaproterenol (Meta) (107 M)- or phenylephrine (Pheny) (106 M)-stimulated hepatocyte proliferation in presence of HGF. Hepatocytes were cultured with HGF (5 ng/ml) at various plating densities for 4 h and 21 h. Data are expressed as mean ± S.E. of three separate experiments. *P < .05, **P < .01 compared with respective controls.

Dose-dependent effects of metaproterenol and phenylephrine on HGF-stimulated hepatocyte DNA synthesis and proliferation during the early and late phases of primary culture. To determine the influence of alpha and beta adrenergic mechanisms on the HGF action, we examined the dose-dependent effects of phenylephrine and metaproterenol on HGF-stimulated DNA synthesis and proliferation at a low density during the early and late phases of culture (table 1). Metaproterenol alone had almost no effect on hepatocyte DNA synthesis and proliferation in the range of 10⁸ to 10⁶ M (data not shown). However, the ability of HGF to induce hepatocyte DNA synthesis and proliferation was significantly potentiated by the addition of metaproterenol with the maximal effect seen at a concentration of 10⁷ M. The potentiation was dose-dependent for metaproterenol up to 10⁷ M and showed EC₅₀ values of 45 ± 6.0 nM (DNA synthesis; n = 3) and 60 ± 5.2 nM (nucleus number; n = 3). Phenylephrine alone had almost no effect on hepatocyte DNA synthesis and proliferation in the range of 10⁶ to 10⁵ M (data not shown). In contrast, the ability of HGF to induce hepatocyte DNA synthesis and proliferation was significantly potentiated by the addition of phenylephrine with the maximal effect seen at a concentration of 10⁶ M. The potentiation was dose-dependent for phenylephrine up to 10⁶ M and showed EC₅₀ values of 250 ±32 nM (DNA synthesis; n = 3) and 600 ± 51 nM (nucleus number; n = 3).

Effects of selective beta-1 and beta-2 adrenergic blockers on metaproterenol-stimulated hepatocyte DNA synthesis and proliferation in the presence of HGF. Beta Adrenergic receptors consist of beta-1 and beta-2 subtypes. Therefore, to further confirm beta-2 adrenergic receptor mediation of metaproterenol-stimulated hepatocyte DNA synthesis and proliferation in the presence of 5 ng/ml HGF, we examined the effects of a specific beta-1 adrenergic blocker, metoprolol, and a specific beta-2 adrenergic blocker, butoxamine, on the potentiation of the HGF effects induced by metaproterenol. As shown in table 2, the effects of metaproterenol were clearly mediated via the beta-2 adrenergic receptor, because the beta-2 selective blocker, butoxamine (10⁶ M), completely inhibited the metaproterenol effect whereas the beta-1 selective blocker, metoprolol (10⁶ M), had no effect on HGF potentiation during any phase of the primary culture. Metoprolol and butoxamine alone had no direct effects on HGF-stimulated hepatocyte DNA synthesis and proliferation. However, metaproterenol-stimulated hepatocyte DNA synthesis was completely blocked by a nonspecific beta adrenergic blocker, propranolol (10⁶ M), without affecting the HGF response. In addition, stimulation of hepatocyte DNA synthesis and proliferation was not observed with the addition of a beta-1 selective agonist, dobutamine (10⁷-10⁵ M), which indicates that potentiation of the HGF effects by metaproterenol is mediated mainly through the beta-2 adrenergic receptors.

View this table: [in this window] [in a new window]   TABLE 2 Effects of specific beta-1 and beta-2 adrenergic antagonists, UK-14304 and H-89 on metaproterenol- and dibutyryl cAMP-stimulated hepatocyte DNA synthesis and proliferation in the presence of HGF Hepatocytes were plated at a density of 3.3 × 104 cells/cm2 and cultured. Specific beta-1 and beta-2 adrenergic antagonists, UK-14304, H-89, metaproterenol and db-cAMP were added with 5 ng/ml HGF immediately after medium change and cells were cultured for a further 4 h and 21 h. Concentrations were as follows: metaproterenol, 107 M; propranolol, 105 M; metoprolol, 106 M; butoxamine, 106 M; dobutamine, 106 M; UK-14304, 106 M; db-cAMP, 107 M; H-89, 107 M. Values are expressed as mean ± S.E. from three independent preparations. Control values for hepatocyte DNA synthesis and the number of nuclei were 573.7 ± 60.2 dpm/mg protein/h and 3.54 ± 0.15 × 104 nuclei/cm2, respectively. Significant differences from HGF alone are indicated by * P < .05.

Effects of H-89 and UK-14304 on metaproterenol- and db-cAMP-stimulated hepatocyte DNA synthesis and proliferation in the presence of HGF. We previously showed that during culture of adult rat hepatocytes, which show a very low alpha-2 and beta adrenergic response in vivo, these responses increase rapidly as a result of the addition of insulin or EGF (Ogihara, 1995, 1996a, b). Based on these findings, we examined the influence of UK14304 (Cambridge, 1981), an alpha-2 adrenergic agonist, on metaproterenol- and db-cAMP-stimulated hepatocyte DNA synthesis and proliferation in the presence 5 ng/ml HGF. As shown in table 2, we found that db-cAMP (10⁷ M) also potentiates hepatocyte DNA synthesis and proliferation induced by HGF. UK14304 (10⁶ M) inhibited hepatocyte DNA synthesis caused by 10⁷ M metaproterenol in the presence of 5 ng/ml HGF. In contrast, UK14304 did not affect db-cAMP-stimulated hepatocyte DNA synthesis and proliferation in the presence of HGF. The ability of UK14304 to inhibit metaproterenol-stimulated hepatocyte DNA synthesis was blocked by yohimbine (10⁵ M; not shown). Each agent alone had no direct effect on either hepatocyte DNA synthesis or proliferation in primary culture.

7

7

Effects of specific alpha-1 and alpha-2 adrenergic antagonists on the phenylephrine-stimulated hepatocyte DNA synthesis and proliferation in the presence of HGF. Alpha adrenergic receptors consist of alpha-1 and alpha-2 subtypes. Therefore, to further confirm alpha-1 adrenergic receptor mediation of phenylephrine-stimulated hepatocyte DNA synthesis and proliferation in the presence of 5 ng/ml HGF, we examined the effects of a specific alpha-1 adrenergic blocker, prazosin, and a specific alpha-2 adrenergic blocker, yohimbine, on the phenylephrine potentiation of the HGF effects. As shown in table 3, the effects of phenylephrine were clearly mediated via the alpha-1 adrenergic receptors, because the alpha-1 selective blocker, prazosin (10⁶ M), completely inhibited the phenylephrine effect, whereas the alpha-2 selective blocker, yohimbine (10⁶ M), had no effect on phenylephrine action during any phase of the primary culture. Prazosin and yohimbine alone had no direct effects on HGF-stimulated hepatocyte DNA synthesis or proliferation. UK-14304 did not affect phenylephrine-induced hepatocyte DNA synthesis and proliferation, which suggests that alpha-2 receptor-mediated mechanism does not couple to PLC.

View this table: [in this window] [in a new window]   TABLE 3 Effects of specific alpha-1 and alpha-2 adrenergic antagonists, U-73122, sphingosine and ionomycin on phenylephrine- and phorbol ester-stimulated hepatocyte DNA synthesis and proliferation in the presence of HGF Hepatocytes were plated at a density of 3.3 × 104 cells/cm2 and cultured. Specific alpha-1 and alpha-2 adrenergic antagonists, sphingosine, ionomycin, phenylephrine and PMA were added with 5 ng/ml HGF immediately after medium change, and cells were cultured for a further 4 h and 21 h. Concentrations were as follows: phenylephrine, 106 M; prazosin, 106 M; yohimbine, 106 M; UK-14304, 106 M; U-73122,a 106 M; U-73122,b 3 × 106 M; sphingosine,a 3 × 106 M; sphingosine,b 105 M; ionomycin, 105 M; PMA, 107 M. Values are expressed as mean ± S.E. from three independent preparations. Control values for hepatocyte DNA synthesis and the number of nuclei were 546.2 ± 63.2 dpm/mg protein/h and 3.48 ± 0.11 × 104 nuclei/cm2, respectively. Significant differences from HGF alone are indicated by * P < .05, ** P < .01.

Effects of U-73122, sphingosine, PMA and ionomycin on the HGF-stimulated hepatocyte DNA synthesis and proliferation in the absence or presence of phenylephrine. We investigated the role of PLC and its intracellular second messengers (i.e., DG and calcium ion) on the HGF-stimulated hepatocyte DNA synthesis and proliferation in the absence or presence of phenylephrine. As shown in table 3, a PLC- inhibitor, U-73122 (Thompson et al., 1991), attenuated HGF action on hepatocyte DNA synthesis and proliferation in the absence or presence of phenylephrine. To elucidate whether or not DG, a direct activator of PKC, is involved in the HGF-stimulated hepatocyte DNA synthesis and proliferation, hepatocytes were treated with PMA, a synthetic analog of DG (Castagna et al., 1982), for 4 h and 21 h. PMA (10⁷ M) alone had no significant effect on hepatocyte DNA synthesis and proliferation, but did potentiate the ability of HGF to stimulate hepatocyte DNA synthesis and proliferation. In addition, U73122 (3 × 10⁶ M) attenuated phenylephrine but not PMA action on hepatocyte DNA synthesis and proliferation induced by HGF. Pretreatment of hepatocytes with a PKC inhibitor, sphingosine (10⁶ M), partially prevented, whereas a higher concentration of sphingosine (3 × 10⁶ M) significantly blocked the HGF action on the hepatocyte DNA synthesis and proliferation in the absence or presence of phenylephrine at early and late phases of culture. Each agent alone had no direct effect on either hepatocyte DNA synthesis or proliferation in primary culture. Similarly, to determine the possible involvement of intracellular calcium mobilization in hepatocyte DNA synthesis and proliferation, cells were cultured with 10⁵ M calcium ionophore (Xiaomei et al., 1995), ionomycin, for 4 h and 21 h. No changes in hepatocyte DNA synthesis and proliferation were observed with the dose of ionomycin. In addition, other calcium-mobilizing agents such as angiotensin II and arginine vasopressin (10⁸-10⁶ M) did not affect hepatocyte DNA synthesis and proliferation induced by HGF (data not shown).

Effect of specific inhibitors of signal-transducing enzymes on hepatocyte DNA synthesis and proliferation induced by HGF with or without agents that elevate cAMP. We investigated whether or not the mitogenic responses of hepatocytes to HGF alone and HGF with metaproterenol or phenylephrine are mediated by signal transducers such as receptor tyrosine kinase, PI(3)K or p70 S6K. To determine whether or not HGF-stimulated DNA synthesis and proliferation requires receptor tyrosine kinase activity, hepatocytes were treated with HGF in the presence and absence of a specific tyrosine kinase inhibitor, genistein (Akiyama et al., 1987), for 4 h and 21 h. As shown in table 4, genistein almost completely blocked HGF-induced stimulation of hepatocyte DNA synthesis and the proliferative effects of HGF with or without metaproterenol. Treatment of hepatocytes with a specific PI(3)K inhibitor, wortmannin (10⁷ M) (Baggiolini et al., 1987; Dewald et al., 1988; Sanchez-Margalet et al., 1994; Ui et al., 1995), also completely inhibited HGF-induced stimulation of hepatocyte DNA synthesis and proliferation in the absence or presence of metaproterenol or phenylephrine. Table 4 also shows that the immunosuppressant, rapamycin (10 ng/ml) (Chung et al., 1992; Price et al., 1992; Downward, 1994), almost completely attenuated both the mitogenic effects of HGF and co-mitogenic effects of metaproterenol and phenylephrine on hepatocyte DNA synthesis and proliferation. The strong mitogenic effects of HGF with metaproterenol or phenylephrine were completely blocked by the addition of a DNA polymerase  inhibitor, aphidicolin (10 µg/ml).

View this table: [in this window] [in a new window]   TABLE 4 Effect of specific inhibitors of signal-transducing elements on hepatocyte DNA synthesis and number of nuclei induced by metaproterenol and phenylephrine in the presence of HGF Hepatocytes were plated at a density of 3.3 × 104 cells/cm2 and cultured. Specific inhibitors of signal-transducing elements were added with 5 ng/ml HGF immediately after medium change, and cells were cultured for a further 4 h and 21 h. Concentrations were as follows: dexamethasone, 1010 M; metaproterenol, 107 M; phenylephrine, 106 M; genistein, 5 × 106 M; aphidicolin, 10 µg/ml; wortmannin, 107 M; rapamycin, 10 ng/ml. Values are expressed as mean ± S.E. from three independent preparations. Control values for hepatocyte DNA synthesis and the number of nuclei were 533.6 ± 59.7 dpm/mg protein/h and 3.62 ± 0.14 × 104 nuclei/cm2, respectively. Significant differences from HGF alone are indicated by * P < .05, ** P < .01, *** P < .001.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

We demonstrated that hepatocyte DNA synthesis and proliferation in the primary culture were stimulated 3 to 4 h after the addition of HGF (fig. 1). The mechanisms by which HGF rapidly stimulated hepatocyte DNA synthesis and proliferation may be dependent on dexamethasone in the culture medium, because the rapid stimulatory effects of HGF are dose-dependently inhibited by increasing concentrations of the hormone (fig. 3). The glucocorticoids, dexamethasone and hydrocortisone, have been shown to improve the plating efficiency and viability of hepatocytes, and they are used routinely in primary cultures of adult rat hepatocytes. Some investigators, including the authors, remove the glucocorticoids after an initial attachment period (Ichihara et al., 1980), whereas others maintain the cells in the presence of glucocorticoids during the entire growth stimulatory period (Richman et al., 1976). In addition, they have used relatively large doses of the glucocorticoids (10⁸-10⁶ M) in their cultures. Accordingly, the addition of a low concentration of dexamethasone (i.e., 10¹⁰ M) may explain why the results obtained in our short-term studies were different from those of previous extensive studies with longer term culture.

The ability of HGF (5 ng/ml) to induce hepatocyte DNA synthesis and proliferation is strictly dependent on the initial plating density in the presence or absence of metaproterenol (figs. 5 and 6) or phenylephrine (figs. 5 and 6). The mechanisms associated with the density dependence of hepatocyte DNA synthesis and proliferation probably involve cell-to-cell contact (Nakamura et al., 1983a, 1984; Kajiyama and Ui, 1994) and/or the production of inhibitory autocrine factor(s) by hepatocytes in primary culture (Nakamura et al., 1983a). However, further studies are required to confirm this hypothesis.

We have previously demonstrated that cAMP and cAMP-dependent protein kinase (PKA) modulate the regulation of hepatocyte DNA synthesis and proliferation in the presence of EGF (Kimura and Ogihara, 1997a) or insulin (Kimura and Ogihara, in press, 1997b): this conclusion was based on the finding that effects of extracellular application of the cell-permeable cAMP analog, db-cAMP, which directly activates PKA, or the indirect adenylate cyclase activator, metaproterenol, are almost completely blocked by a specific PKA inhibitor, H-89 (Zuscik et al., 1994). In the present study, hepatocyte proliferation stimulated by metaproterenol or db-cAMP in the presence of HGF was also inhibited by the PKA inhibitor, H-89, suggesting the involvement of PKA (table 2). In addition, the notion of membrane adenylate cyclase involvement is supported by the inhibitory effect of a specific alpha-2 adrenergic agonist, UK-14304 (Cambridge, 1981), on metaproterenol-stimulated, but not db-cAMP-stimulated hepatocyte DNA synthesis and proliferation in the presence of HGF. These results can likely be attributed to activation of the adenylate cyclase/PKA pathway. However, the role of the second messenger, cAMP, in the control of hepatocyte DNA synthesis and proliferation remains controversial. Cyclic AMP can either stimulate or inhibit DNA synthesis depending on the culture conditions (Bronstad and Christoffersen, 1980; Bronstad et al., 1983; Mahler and Wilce, 1988; Vintermyr et al., 1989; Refsnes et al., 1992). For example, elevated hepatocyte cAMP levels have been reported to inhibit HGF-stimulated DNA synthesis and proliferation (Marker et al., 1992). In contrast, our results showed that the proliferative effects of HGF are likely, at least in part, to depend on cAMP. Presently, the biological mechanisms by which cAMP modulates hepatocyte DNA synthesis and proliferation remain to be elucidated.

HGF reportedly acts through tyrosine kinase receptors that phosphorylate and activate PLC, which leads to enhanced DG and IP₃ production and mobilization of calcium from intracellular stores (Berridge, 1993; Xiaomei et al., 1995). Alpha-1 adrenergic agonists, such as phenylephrine and norepinephrine, exert their action through the activation of PLC-. The mechanisms leading to stimulation of hepatocyte DNA synthesis and proliferation by HGF in the absence or presence of phenylephrine have been investigated (table 3) with two mechanistically distinct inhibitors of signal transducers, U73122 (Thompson et al., 1991) and sphingosine (Merrill et al., 1989). The PLC- inhibitor, U73122 (3 × 10⁶ M), and the PKC inhibitor, sphingosine (3 × 10⁶ M), attenuated both mitogenic effects of HGF and co-mitogenic effects of phenylephrine, which suggests that PLC- and PKC play an important role in the HGF regulation of hepatocyte DNA synthesis and proliferation. This was supported further by the findings that PMA, a synthetic analog of DG, markedly potentiated the effects of HGF on hepatocyte DNA synthesis and proliferation, and that U73122 attenuated phenylephrine, but not PMA effects on hepatocyte DNA synthesis and proliferation. In agreement with these findings, tyrosine kinase receptors, such as EGF and PDGF, are known to generate IP₃ and DG by interacting directly with PLC- to stimulate hepatocyte growth and proliferation (Ullrich and Schlessinger, 1990; Cantley et al., 1991). On the other hand, if the effects of HGF in the absence or presence of phenylephrine were mediated through calcium, the calcium would be replaced by calcium ionophore, ionomycin (Xiaomei et al., 1995). However, calcium ions did not appear to be involved in the HGF effects in the absence or presence of phenylephrine, because no changes in hepatocyte DNA synthesis and proliferation were observed when cells were cultured with 10⁵ M ionomycin for 4 h and 21 h (table 3). In addition, other calcium-mobilizing agents, such as angiotensin II and arginine vasopressin (10⁸-10⁶ M), did not affect hepatocyte DNA synthesis and proliferation induced by HGF in the absence or presence of phenylephrine (data not shown). Therefore, HGF effects can likely be attributed to activation of the PLC/PKC pathway.

To investigate the possible mechanisms involved in the activation of hepatocyte DNA synthesis and proliferation induced by HGF in primary cultures of adult rat hepatocytes, hepatocytes were cultured with specific inhibitors of signal transducers (table 4). Hepatocyte DNA synthesis and proliferation induced by HGF was almost completely blocked by specific inhibitors of signal transducers, such as a specific tyrosine kinase inhibitor, genistein (Akiyama et al., 1987), a specific PI(3)K inhibitor, wortmannin (Baggiolini et al., 1987; Dewald et al., 1988; Sanchez-Margalet et al., 1994; Ui et al., 1995), and a p70 S6K inhibitor, rapamycin (Chung et al., 1992; Price et al., 1992; Downward, 1994). These results suggest that these signal transducers play an essential role in the mitogenic activity induced by HGF.

In conclusion, the present results demonstrate for the first time that HGF can rapidly induce the proliferation of adult rat hepatocytes in a primary culture. This induction is dependent on the initial plating density. The present results also suggest that after stimulation with HGF, activation of tyrosine kinase, PI(3)K, PLC and P70 S6K is essential for hepatocyte DNA synthesis and proliferation. The mitogenic effects of HGF were potentiated by both a beta-2 adrenergic agonist, metaproterenol, which is mediated primarily through PKA and an alpha-1 adrenergic agonist, phenylephrine, which is mediated primarily through PKC. Thus, both alpha-1 and beta-2 adrenergic action may have a positive influence, whereas alpha-2 adrenergic action may negatively influence normal liver growth and liver regeneration induced by HGF in vivo.