Transgenic Expression of Dominant Negative Tuberin through a Strong Constitutive Promoter Results in a Tissue-specific Tuberous Sclerosis Phenotype in the Skin and Brain

Baskaran Govindarajan,Daniel J Brat,Marie Csete,William D Martin,Emma Murad,Karin Litani,Cynthia Cohen,Francesca Cerimele,Matthew Nunnelley,Benjamin Lefkove,Toshiyuki Yamamoto,Chunsik Lee,Jack L Arbiser

doi:10.1074/jbc.m411768200

Baskaran Govindarajan, Daniel J Brat + Show 11 more

Open Access

https://doi.org/10.1074/jbc.m411768200

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Abstract

Tuberous sclerosis (TS) is a common autosomal dominant disorder caused by loss or malfunction of hamartin (tsc1) or tuberin (tsc2). Many lesions in TS do not demonstrate loss of heterozygosity for these genes, implying that dominant negative forms of these genes may account for some hamartomas and neoplasms in TS. To test this hypothesis, we expressed a dominant negative allele of tuberin (DeltaRG) behind the cytomegalovirus promoter in NIH3T3 cells and transgenic mice. This allele binds hamartin but has a deletion in the C terminus of tuberin, leading to constitutive activation of rap1 and rab5/rabaptin. Expression of DeltaRG in NIH3T3 cells led to a strong induction of reactive oxygen species, induction of vascular endothelial growth factor, and malignant transformation in vivo. Expression of DeltaRG driven by the constitutive cytomegalovirus promoter led to high level expression in all murine tissues examined, including skin, kidney, liver, and brain. Surprisingly, mice expressing the DeltaRG transgene developed a fibrovascular collagenoma in the dermis, which closely resembles the Shagreen patch observed in human patients with TS. In addition, numerous small subpial collections of external granule cells in the cerebellum were observed, which may be the murine equivalent of subependymal giant cell astrocytomas or tubers commonly seen in TS patients. Thus, expression of a dominant negative tuberin in multiple tissues can lead to a tissue-specific phenotype resembling some of the findings in human TS. Our data are the first to demonstrate that specific signaling abnormalities underlie specific hamartomas in a model of a human genetic disorder.

Highlights

Tuberous sclerosis is an autosomal dominant disorder that causes morbidity and mortality because of seizures, mental retardation, hamartomas, and benign and malignant neoplasms [1,2,3]
We study the effect of a tuberin mutant [12] in a transgenic mouse when the transgene is expressed behind the cytomegalovirus promoter
We have shown previously that PDGF-BB plays an important role in the pathogenesis of tuberous sclerosis lesions

Summary

Introduction

Tuberous sclerosis is an autosomal dominant disorder that causes morbidity and mortality because of seizures, mental retardation, hamartomas, and benign and malignant neoplasms [1,2,3]. Whereas neoplasms occur commonly in these mice, hamartomas have not been described [8, 9]. These hamartomas include human skin lesions, including collagenomas and angiofibromas as well as certain brain lesions such as the abnormalities in neural migration that result in tubers and subependymal giant cell astrocytomas. We demonstrate that dominant negative tuberin can induce signaling abnormalities in cultured cells and induce development of hamartomas in transgenic mice. These hamartomas occur in a tissue-specific manner even when the transgene is ubiquitously expressed in all tissues. Endothelial growth factor; LOH, loss of heterozygosity; PDGF, plateletderived growth factor; DCF, dichlorofluorescein; PBS, phosphate-buffered saline

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