The proximal islet-specific glucose-6-phosphatase catalytic subunit-related protein autoantigen promoter is sufficient to initiate but not maintain transgene expression in mouse islets in vivo.

We have previously reported the discovery of an islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) that is predominantly expressed in islet beta-cells. IGRP has recently been identified as a major autoantigen in a mouse model of type 1 diabetes. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion gene expression in transiently transfected islet-derived hamster insulinoma tumor and betaTC-3 cells revealed that the promoter region located between -306 and +3 confers high-level reporter gene expression. To determine whether this same promoter region is sufficient to confer islet beta-cell-specific gene expression in vivo, it was ligated to a beta-galactosidase reporter gene, and transgenic mice expressing the resulting fusion gene were generated. In two independent founder lines, this -306 to +3 promoter region was sufficient to drive beta-galactosidase expression in newborn mouse islets, predominantly in beta-cells, which was initiated during the expected time in development, around embryonic day 12.5. However, unlike the endogenous IGRP gene, beta-galactosidase expression was also detected in the cerebellum. Moreover, beta-galactosidase expression was almost completely absent in adult mouse islets, suggesting that cis-acting elements elsewhere in the IGRP gene are required for determining appropriate IGRP tissue-specific expression and for the maintenance of IGRP gene expression in adult mice.

A polymorphic form of steroidogenic factor-1 is associated with adrenocorticotropin resistance in y1 mouse adrenocortical tumor cell mutants.

ACTH resistance in mutant derivatives of the Y1 mouse adrenocortical tumor cell line results from a defect that affects the activity of steroidogenic factor-1 (SF1), thereby preventing the expression of the melanocortin-2 receptor. In this report, we show that the SF1 genes in ACTH-resistant mutants differ from the gene in ACTH-responsive Y1 cells by two base changes-one that changes an Ala to Ser at codon 172, and one in the third position of codon 3 that does not affect the protein sequence. Furthermore, several of the mutants contain multiple copies of this alternate SF1 gene (SF1(S172)) on acentric chromosome fragments. The SF1(S172) allele represents a polymorphism rather than a spontaneous mutation because the two SF1 alleles can be traced to the hybrid mouse strain (C57L/J x A/HeJ) from which the original adrenal tumor was derived. The SF1(A172) allele also is found in C57Bl/6J and C57Bl/10J mice, whereas the SF1(S172) allele also is found in C3H/HeJ and DBA/2J mice. The two forms of SF1 had only modest differences in activity suggesting that the SF1 polymorphism per se is not directly responsible for ACTH resistance. Our results indicate that the SF1(S172) allele is a marker of ACTH resistance in this family of adrenocortical tumor cells.

A polymorphic form of steroidogenic factor 1 associated with ACTH receptor deficiency in mouse adrenal cell mutants.

We have described a family of adrenocortical tumor cell mutants (including clones OS3, Y6, and 10r9) that are resistant to ACTH because they fail to express the gene encoding the ACTH receptor (MC2R). The MC2R deficiency results from a mutation that impairs the activity of the nuclear receptor steroidogenic factor 1 (SF1) at the MC2R promoter. In this report, we show that ACTH resistance in the mutant clones is associated with a Sf1 gene that has Ser at codon 172 instead of Ala. In two of the three mutant clones, this Sf1 allele is amplified together with flanking DNA from chromosome 2 that includes the genes encoding germ cell nuclear factor and the beta-type proteosome subunit Psmb7. SF1(A172) and SF1(S172) exhibit little or no difference in transcriptional activity in SF1-dependent reporter gene assays, suggesting that SF1(S172) per se is not directly responsible for the loss of MC2R expression. Instead, the Sf1(S172) allele appears to be a marker of ACTH resistance in this family of adrenocortical tumor cell mutants, possibly reflecting the activity of a neighboring gene.

SF1 polymorphisms in the mouse and steroidogenic potential.

ACTH-resistance in four mutant derivatives of a mouse adrenocortical tumor cell line results from a defect that reduces the activity of steroidogenic factor-1 (SF1) thereby preventing expression of the ACTH receptor and other SF1-dependent genes. The SF1 genes from these mutants contain a sequence difference that changes an Ala to Ser at codon 172. Steroidogenic factor-1(S172) represents a polymorphism rather than a spontaneous mutation since the two forms of SF1, SF1(A172), and SF1(S172), can be traced to the hybrid mouse strain (C57L/J x A/HeJ) from which the original adrenal tumor was derived. The SF1(S172) allele is amplified in three of the four mutant clones together with the neighboring genes germ cell nuclear factor and LIM homeobox2. The two forms of SF1 had only modest differences in transcriptional activity in reporter gene assays, suggesting that the SF1 polymorphism per se is not directly responsible for the loss of mc2r expression. Rather, ACTH resistance in this family of adrenocortical tumor cell mutants may be due to a closely linked gene on the SF1(S172) allele. Mouse strains with reportedly high steroidogenic capacity (C57Bl/6J, C57Bl/10J) also have the SF1(A172) allele while mouse strains with low steroidogenic capacity (C3H/HeJ, DBA/2J) have the SF1(S172) allele. These latter observations suggest that the two SF1 alleles also may be markers of steroidogenic potential among mouse strains.

Steroidogenic factor 1: an essential mediator of endocrine development.

The orphan nuclear receptor steroidogenic factor 1 (SF-1, also called Ad4BP and officially designated NR5A1) has emerged as an essential regulator of endocrine development and function. Initially identified as a tissue-specific transcriptional regulator of the cytochrome P450 steroid hydroxylases, SF-1 has considerably broader roles, as evidenced from studies in knockout mice lacking SF-1. The SF-1-knockout mice lacked adrenal glands and gonads and therefore died from adrenal insufficiency within the first week after birth. In addition, SF-1 knockout mice exhibited male-to-female sex reversal of their internal and external genitalia, impaired expression of multiple markers of pituitary gonadotropes, and agenesis of the ventromedial hypothalamic nucleus (VMH). These studies delineated essential roles of SF-I in regulating endocrine differentiation and function at multiple levels, particularly with respect to reproduction. This chapter will review the experiments that established SF-1 as a pivotal, global determinant of endocrine differentiation and function. We next discuss recent insights into the mechanisms controlling the expression and function of SF-1 as well as the current status of research aimed at delineating its roles in specific tissues. Finally, we highlight areas where additional studies are needed to expand our understanding of SF-1 action.