KIF20A mRNA and its product MKlp2 are increased during hepatocyte proliferation and hepatocarcinogenesis.

Mitotic kinesin-like protein 2 (MKlp2), a microtubule-associated motor, is required during mitosis exit for the final step of cytokinesis. It also contributes to retrograde vesicular trafficking from the Golgi apparatus to the endoplasmic reticulum in interphase. The KIF20A gene encoding MKlp2 is controlled by the E2F-retinoblastoma protein-p16 pathway, and its widely expressed mRNA is found in fetal and proliferating adult tissues. The expression pattern and function of MKlp2 in the adult liver, however, have not been investigated. We report herein that MKlp2 transiently accumulates in vivo during mouse liver regeneration after partial hepatectomy and is strongly overexpressed in preneoplastic and neoplastic mouse liver. In vitro in mitogen-stimulated primary hepatocytes, MKlp2 accumulated in the nucleus during the G2 phase of the cell cycle coincident with the mitotic kinase Aurora B. Human hepatoma cell lines exhibited high levels of MKlp2; however, it was undetectable in normal human hepatocytes. RNAi-mediated MKlp2 knockdown in hepatoma cells induced polyploidization consistent with its essential function in promoting cytokinesis and inhibited cell proliferation without inducing apoptosis. KIF20A mRNA was strongly accumulated in a large series of human hepatocellular carcinomas, with the highest expression observed in tumors with genomic instability. Accumulation of MKlp2 in normal proliferating, preneoplastic, and transformed hepatocytes suggests that MKlp2 contributes to both normal and pathologic hepatocyte proliferation and is linked to tumor aggressiveness in human hepatocellular carcinomas.

Identification of a novel function of PiT1 critical for cell proliferation and independent of its phosphate transport activity.

PiT1 is a Na(+)-phosphate (P(i)) cotransporter located at the plasma membrane that enables P(i) entry into the cell. Its broad tissue expression pattern has led to the idea that together with the closely related family member PiT2, PiT1 is the ubiquitous supplier of P(i) to the cell. Moreover, the role of P(i) in phosphorylation reactions, ATP production, DNA structure, and synthesis has led to the view that P(i) availability could be an important determinant of cell growth. However, these issues have not been clearly addressed to date, and the role of either P(i) or PiT proteins in cell proliferation is unknown. Using RNA interference in HeLa and HepG2 cells, we show that transient or stable PiT1 depletion markedly reduces cell proliferation, delays cell cycle, and impairs mitosis and cytokinesis. In vivo, PiT1 depletion greatly reduced tumor growth when engineered HeLa cells were injected into nude mice. We provide evidence that this effect on cell proliferation is specific to PiT1 and not shared by PiT2 and is not the consequence of impaired membrane Na(+)-P(i) transport. Moreover, we show that modulation of cell proliferation by PiT1 is independent from its transport function because the proliferation of PiT1-depleted cells can be rescued by non-transporting PiT1 mutants. PiT1 depletion leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase, whereas other MAP kinases and downstream targets of mammalian target of rapamycin (mTOR) remain unaffected. This study is the first to describe the effects of a P(i) transporter in cell proliferation, tumor growth, and cell signaling.

The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents.

The formation of polyploid cells is part of the developmental program of several tissues. During postnatal development, binucleated tetraploid cells arise in the liver, caused by failure in cytokinesis. In this report, we have shown that the initiation of cytokinesis failure events and the subsequent appearance of binucleated tetraploid cells are strictly controlled by the suckling-to-weaning transition in rodents. We found that daily light/dark rhythms and carbohydrate intake did not affect liver tetraploidy. In contrast, impairment of insulin signaling drastically reduced the formation of binucleated tetraploid cells, whereas repeated insulin injections promoted the generation of these liver cells. Furthermore, inhibition of Akt activity decreased the number of cytokinesis failure events, possibly through the mammalian target of rapamycin signaling complex 2 (mTORC2), which indicates that the PI3K/Akt pathway lies downstream of the insulin signal to regulate the tetraploidization process. To our knowledge, these results are the first demonstration in a physiological context that insulin signaling through Akt controls a specific cell division program and leads to the physiologic generation of binucleated tetraploid liver cells.

Liver tetraploidization is controlled by a new process of incomplete cytokinesis.

Cytokinesis is precisely controlled in both time and space to ensure equal distribution of the genetic material between daughter cells. Incomplete cytokinesis can be associated with developmental or pathological cell division programs leading to tetraploid progenies. In this study we decipher a new mechanism of incomplete cytokinesis taking place in hepatocytes during post-natal liver growth. This process is initiated in vivo after weaning and is associated with an absence of anaphase cell elongation. In this process, formation of a functional contractile actomyosin ring was never observed; indeed, actin filaments spread out along the cortex were not concentrated to the putative site of furrowing. Recruitment of myosin II to the cortex, controlled by Rho-kinase, was impaired. Astral microtubules failed to contact the equatorial cortex and to deliver their molecular signal, preventing activation of the RhoA pathway. These findings reveal a new developmental cell division program in the liver that prevents cleavage-plane specification.

The intraflagellar transport component IFT88/polaris is a centrosomal protein regulating G1-S transition in non-ciliated cells.

Loss of normal primary cilia function in mammals is linked to proliferative diseases, such as polycystic kidney disease, suggesting a regulatory relationship between cilia and cell cycle. The primary cilium expressed by most mammalian cells is nucleated from the elder centriole of the centrosome. The relationship between centrosome and cilia suggests that these structures share functions and components. We now show that IFT88/polaris, a component of the intraflagellar transport, remains associated to the centrosome in a proliferative state. IFT88/polaris is tightly associated with the centrosome throughout the cell cycle in a microtubule- and dynein-independent manner. IFT88/polaris tetratricopeptide repeat motifs are essential for this localization. Overexpression of IFT88/polaris prevents G1-S transition and induces apoptotic cell death. By contrast, IFT88/polaris depletion induced by RNA interference promotes cell-cycle progression to S, G2, and M phases. Finally, we demonstrate that IFT88/polaris interacts with Che-1, an Rb-binding protein that inhibits the Rb growth suppressing function. We propose that IFT88/polaris, a protein essential for ciliogenesis, is also crucial for G1-S transition in non-ciliated cells.