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 ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor α (TNFα, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFα, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-l-lysine–coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 N17, Cdc42 N17, SEK1−, or JNK1− blunted the abilities of glucose, TNFα, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFα, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110α/p110γ) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110α/p110γ) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.

The molecular mechanism(s) by which tumor cells survive after exposure to ionizing radiation are not fully understood. Exposure of A431 cells to low doses of radiation (1 Gy) caused prolonged activations of the mitogen activated protein (MAP) kinase and stress activated protein (SAP) kinase pathways, and induced p21 Cip-1/WAF1 via a MAP kinase dependent mechanism. In contrast, higher doses of radiation (6 Gy) caused a much weaker activation of the MAP kinase cascade, but a similar degree of SAP kinase cascade activation. In the presence of MAP kinase blockade by the speci®c MEK1 inhibitor (PD98059) the basal activity of the SAP kinase pathway was enhanced twofold, and the ability of a 1 Gy radiation exposure to activate the SAP kinase pathway was increased *sixfold 60 min after irradiation. In the presence of MAP kinase blockade by PD98059 the ability of a single 1 Gy exposure to cause double stranded DNA breaks (TUNEL assay) was enhanced at least threefold over the following 24 ± 48 h. The increase in DNA damage within 48 h was also mirrored by a similar decrease in A431 cell growth as judged by MTT assays over the next 4 ± 8 days following radiation exposure. This report demonstrates that the MAP kinase cascade is a key cytoprotective pathway in A431 human squamous carcinoma cells which is activated in response to clinically used doses of ionizng radiation. Inhibition of this pathway potentiates the ability of low dose radiation exposure to induce cell death in vitro.

In primary rat hepatocytes, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a decrease in DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor (CKI) proteins p21Cip-1/WAF1 and p16INK4a. To evaluate the relative importance of these CKIs in mediating this response, we determined the impact of prolonged MAPK activation on DNA synthesis in primary cultures of hepatocytes derived from mice embryonically deleted (null) for either p21Cip-1/WAF1 or p16INK4a. When MAPK was activated in wild-type mouse hepatocytes for 24 h, via infection with a construct to express an inducible oestrogen receptor-Raf-1 fusion protein (DeltaRaf:ER), the expression of p21Cip-1/WAF1 and p16INK4a CKI proteins increased, cyclin-dependent kinase 2 (cdk2) and cdk4 activities decreased, and DNA synthesis decreased. Inhibition of RhoA GTPase function increased the basal expression of p21Cip-1/WAF1 and p27Kip-1 but not p16INK4a, and enhanced the ability of MAPK signalling to decrease DNA synthesis. Ablation of the expression of CCAATT enhancer-binding protein alpha (C/EBPalpha), but not of the expression of C/EBPbeta, decreased the ability of MAPK signalling to induce p21Cip-1/WAF1. When MAPK was activated in p16INK4a-null hepatocytes for 24 h, the expression of p21Cip-1/WAF1 increased, cdk2 and cdk4 activities decreased and DNA synthesis decreased. In contrast with these findings, prolonged activation of the MAPK pathway in hepatocytes from p21Cip-1/WAF1-null mice enhanced cdk2 and cdk4 activities and caused a large increase in DNA synthesis, despite elevated expression of p16INK4a. Inhibition of RhoA GTPase activity in p21Cip-1/WAF1-null cells partly blunted both the basal levels of DNA synthesis and the ability of prolonged MAPK signalling to increase DNA synthesis. Expression of anti-sense p21Cip-1/WAF1 in either wild-type or p16INK4a-null hepatocytes decreased the ability of prolonged MAPK signalling to increase the expression of p21Cip-1/WAF1, and permitted MAPK signalling to increase both cdk2 and cdk4 activities and DNA synthesis. These results argue that the ability of prolonged MAPK signalling to inhibit DNA synthesis in hepatocytes requires the expression of p21Cip-1/WAF1, and that the increased expression of p16INK4a has a smaller role in the ability of this stimulus to mediate growth arrest. Our results also suggest that RhoA function can modulate DNA synthesis in primary hepatocytes via the expression of p21Cip-1/WAF1 and p27Kip-1.

Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular signaling pathways including a cascade of tyrosine phosphorylations. In many cell types, the cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes tyrosine-phosphorylated in response to adhesion; however, the molecular mechanisms regulating integrin-triggered FAK phosphorylation are not understood. Previous studies have shown that the integrin ␤ 1 , ␤ 3 , and ␤ 5 subunit cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an extracellular reporter. In the present study, ␤ 3 cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin ␤ 3 cytoplasmic domain that regulate its ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of FAK. Analysis of these mutants indicated that structural information in both the membrane-proximal and C-terminal segments of the ␤ 3 cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal segment of the ␤ 3 cytoplasmic domain, the highly conserved NPXY motif was found to be required for the ␤ 3 cytoplasmic domain to trigger FAK phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the ␤ 3 cytoplasmic domain was found to be neither required nor sufficient for this signaling event. We also demonstrate that the serine 752 to proline mutation, known to cause a variant of Glanzmann's thrombasthenia, inhibits the ability of the ␤ 3 cytoplasmic domain to signal FAK phosphorylation, suggesting that a single mutation in the ␤ 3 cytoplasmic domain can inhibit both "inside-out" and "outside-in" integrin signaling.