Islet cell hyperplasia in transgenic mice overexpressing EAT/mcl-1, a bcl-2 related gene

Abstract

EAT/mcl-1 (EAT), a bcl-2 related anti-apoptotic gene, is up-regulated at the early stage of differentiation of human embryonal carcinoma cells; cells which serve as a model for early embryogenesis. We generated transgenic mice for the human EAT gene driven by the EF1α promoter in order to elucidate its functional role in vivo. Histologically, these mice exhibited hyperplasia of Langerhans islet cells; pancreatic cell regions composed of both insulin- and glucagon-producing cells. Furthermore, Bax and Bag-1—possible heterodimeric partners for EAT in the anti-apoptotic process—were up-regulated in islets isolated from the EAT transgenic mice. The insulin tolerance test exhibited no significant difference between the EAT transgenic mice and non-transgenic mice, indicating that islet cell hyperplasia was not due to insulin resistance. In conclusion, EAT transgenic mice exhibit hyperplasia of pancreatic β cells. EAT may inhibit apoptosis of β cells, allowing these cells to circumvent the process of apoptosis until the adult stage.

Introduction

We isolated human EAT/mcl-1 (EAT), a bcl-2 related gene, as a gene up-regulated at an early stage of differentiation of an embryonal carcinoma (EC) cell line designated NCR-G3 (Umezawa et al., 1996). Human EC cell lines serve as model systems for early human embryogenesis based upon their multiple differentiation potential. NCR-G3, derived from a testicular EC, differentiates into multiple lineages, including trophectoderm cells, following exposure to retinoic acid (Hata et al., 1992). Human EAT (hEAT) was originally identified as mcl-1, a gene whose expression is induced during differentiation of myeloid leukemia cells (Kozopas et al., 1993); however, we further established that it is likewise up-regulated during early differentiation of EC cells (Umezawa et al., 1996). Recently, we also cloned a murine orthologue of hEAT (murine EAT, mEAT) (Okita et al., 1998).

The EAT gene is considered to be a member of the bcl-2 related gene family based upon its possession of the bcl-2 homology (BH) domains 1, 2, 3 and 4 (Bingle et al., 2000, Kroemer, 1997, Revilla et al., 1997). Bcl-2-related genes display either positive or negative regulatory effects on apoptosis in vitro and in vivo (Adams and Cory, 1998, Chao and Korsmeyer, 1998, Reed, 1998). Previous studies have established that Bcl-2, Bcl-xL, Bcl-w, Bfl-1 and A1 are anti-apoptotic molecules while Bax, Bak, Bcl-xS, Bad, Bid, Bik and Hrk are pro-apoptotic molecules (Boise et al., 1993, Chittenden et al., 1995, D'Sa-Eipper et al., 1996, Gibson et al., 1996, Inohara et al., 1997, Lin et al., 1996, Wang et al., 1996, Yang et al., 1995a). Recent studies revealed that EAT inhibits apoptosis in vitro and in vivo (Akgul et al., 2000a, Akgul et al., 2000b, Ando et al., 1998, Matsushita et al., 1999, Moulding et al., 2000, Reynolds et al., 1994, Sano et al., 2001, Sano et al., 2000).

The in vivo effects of Bcl-2 related genes have been investigated in transgenic or knock-out mice. Bcl-2 transgenic mice are known to promote cell survival in B cells, T cells and thymocytes (McDonnell et al., 1989, Sentman et al., 1991, Siegel et al., 1992, Strasser et al., 1991). Mice deficient for bcl-2 display increased apoptosis in selected tissues (Veis et al., 1993). These phenotypes reflect the anti-apoptotic functions established for bcl-2 in vitro. On the other hand, bax transgenic mice present with increased apoptosis in T cells and mice deficient for bax demonstrate hyperplasia of thymocytes and B cells (Brady et al., 1996a, Brady et al., 1996b, Knudson et al., 1995). Male Bax-deficient mice are infertile with atrophic adult testes and an empty epididymis and vas deferens; a complete cessation of mature sperm cell production occurs in these mice (Knudson et al., 1995). These phenotypes result from the pro-apoptotic functions of Bax.

We generated three lines of EAT transgenic mice, driven by the EF1α promoter, to investigate in vivo the role of EAT during development. Our aim was to determine whether overexpression of the hEAT gene would cause anti-apoptotic effects in vivo similar to the bcl-2 gene. In this report, we show that EAT transgenic mice develop islet cell hyperplasia reflecting inhibition of apoptosis of β cells.