[Cell Cycle 2:2, 96-98, March/April 2003]; © 2003 Landes Bioscience

Spotlight on Cardiology

Regulating Heart Development The Role of Nf1 ABSTRACT

Departments of Medicine and Cell and Developmental Biology; University of Pennsylvania Health System; Philadelphia, Pennsylvania USA *Correspondence to: 954 BRB II; 421 Curie Blvd.; Philadelphia, Pennsylvania 19104 USA; Tel.: 215.898.8731; Fax: 215.573.2094; Email: [email protected]

Neurofibromatosis type 1 (NF1) is one of the most common human genetic disorders and is associated with significant morbidity and mortality. The gene responsible for this disorder, NF1, encodes neurofibromin, which can function to down-regulate ras activity. Mutations that inactivate NF1 result in elevated levels of ras signaling and increased cell proliferation in some tissues. NF1 functions as a tumor suppressor gene; patients inherit one mutated copy and are believed to acquire a “second hit” in tissues that go on to form benign or malignant tumors.1,2 NF1 is expressed widely, yet certain tissues are more susceptible to growth dysregulation in NF1 patients. Cardiovascular defects also contribute to NF1, though the cause remains unclear. In a recent study, we used tissue-specific gene inactivation in mice to study the role of neurofibromin in heart development. A further understanding of neurofibromin function will help to elucidate the pathophysiology of NF1 and will also lead to a better understanding of cell cycle regulation and ras pathways in specific cell types. Finally, we comment on how similar genetic strategies can be used in mice to study the role of additional signaling pathways involved in heart development.

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Received 02/11/03; Accepted 02/12/03

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Aaron D. Gitler Jonathan A. Epstein*

KEY WORDS

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Heart development, Neurofibromatosis, Endothelium, Neural crest, cre,ras, NFAT

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Previously published online as a Cell Cycle “Paper In Press” at http://www.landesbioscience.com/journals/cc/

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MOUSE MODELS OF NF1

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Mice with mutations in the murine Nf1 homologue develop tumors like their human counterparts.3 However, these occur only slowly and sporadically. Chimeric mice composed of cells completely lacking neurofibromin and wild type cells develop tumors that mimic those seen in humans including those of neural crest origin.4 Strains of mice that have been engineered to have linked mutations in both Nf1 and the tumor suppressor p53 also develop tumors characteristic of NF1.4,5 However, mice completely lacking Nf1 do not survive gestation and display dramatic abnormalities of cardiovascular development.3,6,7

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DISSECTING TISSUE-SPECIFIC FUNCTIONS OF Nf1 USING A CRE/LOXP APPROACH Recently, our laboratory collaborated with that of Luis Parada to utilize a Cre/loxP approach in order to determine the cell types in which Nf1 functions during embryonic development.8 The Parada laboratory has developed a floxed allele of Nf1 allowing for tissue-specific inactivation of the gene.9 Since tumors in human patients are generally of neural crest origin, it is not surprising that inactivation of this gene in neural crest cells leads to abnormal proliferation of neural crest derived tissues. We noted hyperplasia of several neural crest derived structures, including the adrenal gland and sympathetic ganglia. Neural crest cells also contribute to the heart, but interestingly inactivation of Nf1 in neural crest cells does not cause cardiovascular defects. Although the heart muscle is not developed normally in embryos completely lacking Nf1, we find that inactivation of Nf1 specifically in myocardial cells has no effect. Surprisingly, cardiovascular defects are produced when Nf1 is inactivated in endothelial cells. Endocardial cushions, the endothelialderived precursors of heart valves, show an overabundance of mesenchymal cells, similar to that seen in Nf1 nulls. We observe malalignment of the outflow tract; previously thought to be a neural crest related defect. The heart muscle is also poorly developed, presumably because signals from endothelium are required for myocardial cell development. However, the cardiovascular defects are not as severe as those seen in complete nulls. While Nf1 deficiency is lethal at midgestation, we have noted some endothelial-specific Nf1 mutants that survive gestation. This could be due to incomplete inactivation of Nf1 in endothelium due to inefficient cre-mediated recombination, or it could relate to altered Cell Cycle

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genetic backgrounds secondary to the multiple crosses required for these experiments. However, it remains possible that the functions of Nf1 in non-endothelial cells contribute to the embryonic cardiac phenotype.

DYSREGULATED RAS SIGNALING IN Nf1-DEFICICENT ENDOTHELIAL CELLS The developing embryo provides a model for the further study of neurofibromin and ras function in endothelium. We find elevated levels of phosphorylated MAPK (mitogen activated protein kinase) in endocardial cells suggesting increased ras signaling in Nf1 mutants. Further, we note precocious nuclear localization of the transcription factor NFATc1 (Nuclear Factor of Activated T cells). NFATc1 activity is required for cardiac development and heart valve formation10,11 and ras activity can modulate NFATc1 nuclear localization in other cell types.12,13 Cytoplasmic NFATc1 is phosphorylated, and dephosphorylation by the phosphatase calcineurin leads to its accumulation in the nucleus. However, our studies do not determine whether alterations in ras activity affect NFATc1 nuclear localization through a calcineurin dependent or independent pathway. Future studies will be aimed at addressing this question, since calcineurin-dependence may offer possible therapeutic options. Calcineurin is inhibited pharmacologically by the immunomodulator cyclosporin A (CsA). It will be interesting to test whether treatment of embryos with CsA is effective in modulating the cardiovascular phenotypes we observe in Nf1 mutants. Furthermore, it remains to be determined if neurofibromin-deficiency in other cell types leads to an increase in nuclear localization of NFATc1 or other NFAT isoforms. While we have demonstrated a correlation between Nf1 deficiency, ras activation, and NFATc1 nuclear localization, we have not determined which, if any, of these downstream effects is required for the development of cell growth abnormalities in the heart. This will be an important next step in working out the details of how downstream mediators of ras signaling are utilized in a tissue-specific manner in the heart. Our identification of a ras" NFATc1 connection in endothelial cells is just one piece to the puzzle. It is likely that effects of ras activation unrelated to NFATc1 contribute to endothelial cell abnormalities in these embryos. Furthermore, neurofibromin may be required to regulate other cellular signaling pathways in addition to the ras pathway.

USING MOUSE MODELS TO STUDY SIGNALING PATHWAYS INVOLVED IN ENDOCARDIAL CUSHION MORPHOGENESIS The endocardial cushion phenotypes of the Nf1 and NFATc1 knockout mice led us to test whether there was altered NFATc1 activity in neurofibromin-deficient endothelial cells in the heart. A growing number of animal models with cardiovascular defects involving the endocardial cushions are emerging in the literature.10,11,14-17 In Figure 1 we speculate as to how some of these gene products may interact in endothelium to regulate endocardial cushion formation. It will be possible to test the relationships between these gene products by intercrossing specific knockout mice. For example, our work suggests that NFATc1 functions downstream of ras and Nf1 in endothelial cells. Does decreased NFATc1 gene dosage alleviate some or all of the cardiovascular defects seen in Nf1-/- embryos? TGFβ signaling has also been implicated in endocardial cushion development.18 Inactivation of Smad6, an intracellular inhibitor of the pathway, leads to hyperplasia of the www.landesbioscience.com

Figure 1. Multiple signaling pathways are involved in endocardial cushion development. A model depicting how the ras signaling pathway may interact with the TGFβ and EGF pathways in endocardial cells. Underlined components indicate an endocardial cushion phenotype when disrupted in mouse. Solid lines indicate experimental evidence for a connection between two components in endothelium and a dashed line represents a hypothetical connection. In this model, loss of Nf1 leads to increased ras activity, which results in translocation of NFATc1 to the nucleus. Elevated ras activity may modulate Smad6-mediated inhibition of the TGFβ pathway. Cardiovascular defects caused by mutations upstream of ras, such as in the EGF receptor may also be due to dysregulated ras signaling.

endocardial cushions and outflow tract defects.17 Crosstalk between Smads and the ras pathway has been demonstrated,19 suggesting that perhaps elevated levels of ras signaling in Nf1-/- hearts leads to increased TGFβ signaling. Again, genetic crosses between Smad6 and Nf1 knockout animals will be useful in determining how these pathways may interact to affect cardiac development. While Smad6 and Nf1 heterozygous mice each have normal cardiovascular development, Smad6;Nf1 compound heterozygotes may exhibit cardiovascular defects, consistent with a genetic interaction.

IMPLICATIONS FOR NF1 PATIENTS Cardiovascular defects, including elevated blood pressure and vascular abnormalities, are associated with NF1. Recent studies suggest an increased incidence of congenital heart disease related to heart valve development, though the incidence remains small.20 These phenotypes may be related to the function of neurofibromin in endothelial cells, though the function of Nf1 in other components of the vasculature including smooth muscle remains to be determined. Just as importantly, the discovery of tissue-specific functions for Nf1 will allow for a further understanding of cell-type specific modulation of ubiquitous cell-signaling pathways like the ras cascade. This understanding will allow for the design of rational therapies to treat tissue-specific pathology in NF1 patients and those with related abnormalities. Acknowledgments JAE is supported by grants from the National Institutes of Health. ADG is supported by a National Institutes of Health Predoctoral Training Grant to the University of Pennsylvania Medical School.

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