Thyroid status affects rat liver regeneration after partial hepatectomy by regulating cell cycle and apoptosis proteins

In rats, various growth factors and hormones, as well as partial hepatectomy (PH) are able to trigger the proliferative response of hepatocytes. Although recent evidence highlights the important role of thyroid hormones and thyroid status in regulating the growth of liver cells in vitro and in vivo models, the mechanism involved in the pro-proliferative effects of thyroid hormones is still unclear. Here we have investigated how in rats made hypo- and hyperthyroid after prolonged treatment respectively with propylthiouracil (PTU) and triiodothyronine (T3), the thyroid status affects liver regeneration after PH by regulating cell cycle and apoptosis proteins. Our results show that both in control and partially hepatectomized animals hyperthyroidism increases the cyclin D1, E and A levels and the activity of cyclin-cdk complexes, and decreases the levels of cdk inhibitors such as p16 and p27. On the contrary hypothyroidism induces a down-regulation of the activity of cyclin cdk complexes decreasing cyclin levels. Thyroid hormones control also p53 and p73, two proteins involved in apoptosis and growth arrest which are induced by PH. In particular, hypothyroidism increases and T3 treatment decreases p73 levels. The analysis of the phosphorylated forms of p42/44 and p38 MAPK revealed that they are induced during hepatic regeneration in euthyroid and hyperthyroid rats whereas they are negatively regulated in hypothyroid rats. In conclusion our data demonstrate that thyroid status can affects liver regeneration, altering the expression and the activity of the proteins involved in the control of cell cycle and growth arrest. Copyright © 2005 S. Karger AG, Basel

Differences between the real and the desired worlds in the results of clinical trials

OBJECTIVE:We refer to the effectiveness (known as pragmatic or real world) and efficacy (known as explanatory or desired or ideal world) of interventions. However, these terms seem to be randomly chosen by investigators who design clinical trials and do not always reflect the true purpose of the study. A pragmatic-explanatory continuum indicator summary tool was thus developed with the aim of identifying the characteristics of clinical trials that distinguish between effectiveness and efficacy issues. We verified whether clinical trials used the criteria proposed by the indicator summary tool, and we categorized these clinical trials according to a new classification.METHOD:A systematic survey of randomized clinical trials was performed. We added a score ranging from 0 (more efficacious) to 10 (more effective) to each domain of the indicator summary tool and proposed the following classifications: high efficacy

Treatment with EGF increases the lenght of S-phase after partial hepatectomy in rat changing the activities of CDKs.

Liver proliferation occurs in the presence of mitogenic stimuli such as partial hepatectomy or growth factors. In this work we investigate how partial hepatectomy and Epidermal Growth Factor (EGF) affect hepatocyte proliferation by modulating cell cycle regulators. EGF administered to non-operated rats increased PCNA (proliferating cell nuclear antigen) expression, whereas when EGF was administered to partially hepatectomized rats it was able to anticipate the increase in PCNA expression to 18h after PH and to prolong it to 34h. Cell cycle progression was examined by monitoring specific markers of late G1- and S-phases. Western blot analysis showed that both treatment with EGF alone and treatment with EGF after PH induce the expression of cyclins D1 and A and of p21(cip1), but inhibites the expression of cyclin E and p27(kip1). EGF administration after PH also significantly affected the activity of the cyclin D1-cdk4 and cyclin E-cdk2 complexes, mainly by changing their time progression: it accelerated the increase in activity to 18h and caused a subsequent drop in activity after 34h; it delayed the activity of the cyclin A-cdk2 complex to 34h. In conclusion we observed that EGF modulates the activity of cdk complexes and induces a different linkage with inhibitory proteins that demonstrates their dual role, depending on their association with different cyclin-cdk complexes

SHORT-TERM EFFECTS OF THYROID HORMONE IN PRENATAL DEVELOPMENT AND CELL DIFFERENTIATION

Extranuclear effects of thyroid hormones, L-T3 and L-T4, normally show a time-course of seconds to minutes, and are probably mediated by specific receptors on the plasma membrane. Since the liver is an important target for thyroid hormones, we have investigated their extranuclear effects on chick embryo hepatocytes at 14 and 19 days of embryonic life. At these stages of their development the hepatocytes show different protein expression patterns and activities and, of particular interest here, different levels of deiodinases. Three essential membrane transport activities were studied: the Na+/H+ exchanger, the amino acid transport System A (AIB transport), and the Na+/K+-ATPase; the signal transduction mechanisms involved were examined. We also focused on the possibility that 3,5-diiodo-L-thyronine (3,5-T2) might represent a new hormone able to mimic the extranuclear effects of thyroid hormones.The Na+/H+ exchanger type 1 (NHE-1) is a ubiquitous plasma membrane integral protein showing both housekeeping functions (modulation of intracellular pH and cell volume) and a regulative role, being activated by growth factors, hormones, integrins and hyperosmolarity. Both L-T3 and L-T4 stimulated the Na+/H+ exchanger over a wide concentration range, giving a bell-shape dose response as already found for other hormones (i.e insulin). The maximum increase of intracellular pH was observed at 1 nM hormone concentration, for both 14 and 19 days old cells, and 3,5-T2 was able to mimic the non genomic effect of thyroid hormones, but less efficiently. A different pattern was found for the Na+-dependent amino acid transport System A, which is highly regulated by hormones, growth factors and amino acid deprivation. At variance with the Na+/H+ exchanger, the activation of AIB transport by thyroid hormones appeared related to the proliferative activity and to the stage of embryonal development, being highly significant at 19 days, when the embryo is close to term, and insensitive to thyroid hormones at the 14 days stage. The effect of 3,5-T2 on System A activity was less evident. Yet another behavior was seen for the third transporter studied; since both L-T3, and its analog 3,5-T2 inhibited the Na+/K+-ATPase in chick embryo hepatocytes at different stages of development, in a dose-dependent way. Signal transduction appears to be mediated by interplay among kinases: PKA, PKC, the MAPK pathway, together with second messengers such as calcium, IP3 and DAG. These data allow us to draw the following conclusions on the role of extranuclear effects of thyroid hormones in cell development and differentiation: First, chick embryo hepatocytes are able to respond to thyroid hormones activating the same transduction pathway as growth factors (i.e. EGF). Second, 3,5-T2 is able to mimic thyroid hormone effects on the three transport systems. This capability of 3,5-T2 might be of physiological relevance as a safety factor, in the case of a possible hypothyroidism in the prenatal period. The results obtained so far indicate that in our experimental system thyroid hormones and 3,5-T2 via a short-term mechanism exert modulating effects on membrane transport activities that are important for development and differentiation. Interestingly all three systems are modulated also by nuclear genomic action

Thyroid hormones regulate DNA synthesis and cell cycle proteins by activation of PKCalpha and p42/44 MAPK in chick embryo hepatocytes.

The molecular mechanism by which thyroid hormones exert their effects on cell growth is still unknown. In this study, we used chick embryo hepatocytes at different stages of development as a model to investigate the effect of the two thyroid hormones, T3 and T4, and of their metabolite T2, on the control of cell proliferation. We observed that T2 provokes increase of DNA-synthesis as well as T3 and T4, independently of developmental stage. We found that this stimulatory effect on the S phase is reverted by specific inhibitors of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (p42/44 MAPK), Ro 31-8220 or PD 98059. Furthermore, the treatment with thyroid hormones induces the activation of PKCalpha and p42/44 MAPK, suggesting their role as possible downstream mediators of cell response mediated by thyroid hormones. The increase of DNA-synthesis is well correlated with the increased levels of cyclin D1 and cdk4 that control the G1 phase, and also with the activities of cell-cycle proteins involved in the G1 to S phase progression, such as cyclin E/A-cdk2 complexes. Interestingly, the activity of cyclin-cdk2 complexes is strongly repressed in the presence of PKC and p42/44 MAPK inhibitors. In conclusion, we demonstrated that the thyroid hormones could modulate different signaling pathways that are able to control cell-cycle progression, mainly during G1/S transitio