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LEK1, a member of the LEK family of proteins, is ubiquitously expressed in developing murine tissues. Our current studies are aimed at identifying the role of LEK1 during cell growth and differentiation. Little is known about the function of LEK proteins. Recent studies in our laboratory have focused on the characterization of the LEK1 atypical Rb-binding domain that is conserved among all LEK proteins. Our findings suggest that LEK1 potentially functions as a universal regulator of pocket protein activity. Pocket proteins exhibit distinct expression patterns during development and function to regulate cell cycle, apoptosis, and tissue-specific gene expression. We show that LEK1 interacts with all three pocket proteins, p107, p130, and pRb. Additionally, this interaction occurs specifically between the LEK1 Rb-binding motif and the "pocket domain" of Rb proteins responsible for Rb association with other targets. Analyses of the effects of disruption of LEK1 protein expression by morpholino oligomers demonstrate that LEK1 depletion decreases cell proliferation, disrupts cell cycle progression, and induces apoptosis. Given its expression in developing cells, its association with pocket proteins, and its effects on proliferation, cell cycle, and viability of cells, we suggest that LEK1 functions in a similar manner to phosphorylation to disrupt association of Rb proteins with appropriate binding targets. Thus, the LEK1/Rb interaction serves to retain cells in a pre-differentiative, actively proliferative state despite the presence of Rb proteins during development. Our data suggest that LEK1 is unique among LEK family members in that it specifically functions during murine development to regulate the activity of Rb proteins during cell division and proliferation. Furthermore, we discuss the distinct possibility that a yet unidentified splice variant of the closely related human CENP-F, serves a similar function to LEK1 in humans.

SUMMARYCENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT) network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.

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A defining characteristic of embryonic cells is their ability to divide rapidly, even in tissues such as cardiac muscle, which cannot divide once fully differentiated. This suggests that regulators of cell division differ in embryonic and differentiated cells. LEK1 is a member of an emerging family of proteins with diverse functions but shared structural domains, including numerous leucine zippers, a nuclear localization site, and a functional Rb-binding domain. LEK1 is expressed ubiquitously in the developing mouse embryo from the earliest stages of differentiation through birth. It is absent in adult tissues, even those that maintain active cell division. We hypothesize that LEK1 is a regulator of mitosis restricted to the developing embryo and early neonate. Here, using BrdU incorporation, we show that LEK1 protein downregulation in cardiac myocytes correlates directly with cessation of DNA synthesis between neonatal days 6 and 10. In contrast, in an immortalized cardiac cell line (HL1 cells), both BrdU incorporation and LEK1 protein expression persist, and actively dividing cells express LEK1. However, BrdU incorporation can be decreased in these cells by treatment with a morpholino targeting LEK1 mRNA. These data suggest a role for LEK1 in regulating the normal embryonic cardiomyocyte cell cycle and in promoting continued mitosis in transformed, abnormally dividing cardiomyocytes.

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Numerous advances in understanding the molecular basis of congenital heart disease have been published in the past year. Highlights are reviewed, focusing on two major topics: genetic syndromes and cardiac organogenesis. Genetic syndromes are discussed in the context of complementary data from targeted mutations in animals and genetic mapping studies in humans. These include the DiGeorge, Holt-Oram, Alagille, familial primary pulmonary hypertension, and Noonan syndromes. Novel concepts in cardiac organogenesis are discussed, including the existence and contribution of an anterior heart field to the developing cardiac outflow tract, novel cell-cell signaling involving migrating neural crest, the origins of the conduction system and initial embryonic heartbeat, and the possibility of a population of cardiac stem cells in the adult heart. The studies reviewed have potential clinical relevance in the near future and will be of interest to the clinician interested in congenital heart disease.