Differential Regulation of the Mitogen-Activated Protein and Stress-Activated Protein Kinase Cascades by Adrenergic Agonists in Quiescent and Regenerating Adult Rat Hepatocytes

Abstract

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the ␣ 1 -adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the ␤ 2 -adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an Partial hepatectomy (PHX) or acute dissociation and primary culturing of hepatocytes trigger their entry into the cell cycle, which is accompanied by alterations in the levels of expression of various liver-specific proteins (14, 34). Epidermal growth factor (EGF), hepatocyte growth factor, and insulin have been shown to induce DNA synthesis in cultured hepatocytes or in quiescent liver (14,29,34). Catecholamines have been shown to increase DNA synthesis in quiescent liver via stimulation of ␣ 1 -adrenergic receptors (ARs) (6,14,18,24,27,34,44,51). The role(s) of ␤ 2 -ARs in the control of hepatic DNA synthesis is less clear, as both stimulatory and inhibitory effects have been described elsewhere (13,15,35). Previous studies have demonstrated that PHX or primary culture of rat hepatocytes significantly increases ␤ 2 -AR, and decreases ␣ 1 -AR, expression and function (24,35,38). However, the capacity of this receptor switch to modulate many of the recently discovered signal transduction pathways, such as the Raf/mitogen-activated protein (MAP) kinase cascade, and its role in the regulation of hepatocyte regeneration are unclear.Raf-1 is a member of a family of serine/threonine protein kinases termed Raf-1, B-Raf, and A-Raf (41). Proto-oncogenes of the Raf family have been implicated in hepatic carcinogenesis (2,11,17,34,40,41). Raf family members function in a signaling cascade that extends from the plasma membrane, via tyrosine kinase (16) and serpentine (22, 36) receptors, to the low-molecular-weight GTP-binding protein Ras. GTP-Ras binds and translocates Raf-1 to the plasma membrane, which leads to the activation of Raf-1 by multiple mechanisms (12,16,30,36). Raf family members in turn activate MEK1/2, which in turn activates the p42/44 MAP kinases, whose activation leads to modulation of downstream transcription factor activities (30, 46). The MAP kinase pathway can be negatively regulated by agonists that elevate cyclic AMP and activate protein kinase A (PK-A), such as glucagon and ␤ 2 -AR agonists (8,28,32,45). Activation of PK-A has been shown to inhibit or delay the act...

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic

“…Male Sprague-Dawley rats (200 g) had access to food and water ad libitum [4]. Anti-Raf-1 (sc-133AC) and anti-B-Raf (sc-166-G) COOH-terminal antibodies, anti-cdk2 (sc-163), anti-p15 INK%b (sc-1429), anti-p16 INK%a (sc-1207), anti-p21 Cip-" (sc-397-G) and anti-p27 Kip-" (sc-527-G) antibodies were from Santa Cruz Biotechnology (Santa Cruz Biotechnologies, CA, U.S.A.), as were anti-Cyclin A-(sc-596), D-(sc-753) and E-(sc-481).…”

“…Glutathione-STransferase (GST)-c-Jun (aal-169) was synthesized in E. coli and purified on Glutathione-Sepharose. Protein preparations of other reagents were as in [4]. The specific MEK1 activation inhibitor PD98059 [29] was from Calbiochem.…”

“…A small (3 cm) vertical mid-line incision was made in the abdominal wall from just below the costal margin\xiphoid process. Hepatocytes were prepared by cannulation of the portal vein and collagenase perfusion of the liver as described [4]. Hepatocytes were placed into primary culture at 2i10& cells per well of a 12 well plate cultured in Dulbecco's modified Eagle's medium (DMEM).…”

“…Partial hepatectomy or acute dissociation by collagenase treatment and primary culture of hepatocytes triggers their entry into the cell cycle [1][2][3][4][5][6][7][8][9][10]. Maximal DNA synthesis in i o occurs 12-36 h post partial hepatectomy and within 5-7 days the liver has regained its original mass [1][2][3].…”

“…Maximal DNA synthesis in i o occurs 12-36 h post partial hepatectomy and within 5-7 days the liver has regained its original mass [1][2][3]. Primary culture in itro in the presence of growth factors such as insulin or epidermal growth factor (EGF) stimulates hepatocyte DNA synthesis which is maximally observed 40-70 h post isolation [1][2][3][4]. Treatment of hepatocytes, isolated from either proliferating or nonproliferating livers, with either insulin or EGF has also been shown to cause an acute phasic increase of mitogen activated protein (MAP) kinase activity with a subsequent stimulation of DNA synthesis [3].…”