Spermatocytes, the most sensitive male germ cells to heatinduced apoptosis, do not respond to hyperthermia by inducing heat shock proteins (HSPs), including HSP70i, which has been previously shown to confer resistance to apoptosis in somatic cells. To dissect the mechanism of heat-induced apoptosis and to determine if we could protect spermatocytes by expressing HSP70i, we engineered transgenic mice that express in spermatocytes constitutively active heat shock transcription factor (HSF)1. Such HSF1 expression did not lead to transcription of inducible Hsp70 genes, but instead induced caspase-dependent apoptosis that mimicked heat shock-induced death of spermatogenic cells. Both mitochondria-dependent and death receptor-dependent pathways appear to be involved in such HSF1-induced apoptosis: the levels of Bcl-2 family proteins became increased, p53 protein accumulated and expression levels of caspase-8 and deathreceptor-interacting proteins (including Fas-associated death domain protein and TNF receptor associated death domain protein) became elevated. Surprisingly, the constitutive spermatocyte-specific expression of HSP70i in doubletransgenic males did not protect against such HSF1-induced apoptosis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with numerous abnormalities recorded at the cellular, molecular, and genetic level. Expression of the basic leucine zipper transcription factor cAMP-responsive element modulator (CREM)␣ was reported to be abnormally increased in T cells from SLE patients. CREM␣ suppresses IL-2 and T cell receptor chain gene transcription by direct binding to the respective promoters. Here, we show that increased CREM expression is the result of enhanced promoter activity. DNA binding analyses reveal direct binding of transcription factor specificity protein-1 (SP-1) to the CREM promoter resulting in enhanced transcriptional activity and increased CREM expression. Protein phosphatase 2A is known to activate SP-1 through dephosphorylation at its serine residue 59. Our results show that nuclei from SLE T cells contain lower levels of Ser 59 -phosphorylated SP-1 protein and a stronger SP-1 binding to the CREM promoter. We conclude that protein phosphatase 2A accounts for enhanced SP-1 dephosphorylation at Ser 59 in SLE T cells. More importantly, CREM promoter activity mirrors reliably disease activity in SLE patients, underscoring its potential role as a biomarker for the prediction of flares in SLE patients. Systemic lupus erythematosus (SLE)3 is a chronic inflammatory disease characterized by an autoimmune process affecting every organ, including joints, kidneys, skin, and the central nervous system (1). T cells have been demonstrated to be important in the pathogenesis of SLE (2). Functional abnormalities in SLE T cells are largely underwritten by altered gene transcription, and recent studies have revealed a vast number of transcription factors in SLE T cells displaying changes in their expression levels and function. Among them, the ␣-isoform of the transcriptional regulator cAMP-responsive element modulator (CREM) has emerged as a potent regulator of target genes that are crucially involved in T cell physiology, e.g. interleukin (IL)-2, T cell receptor chain, and transcription factor c-fos (3-5).The CREM protein family constitutes a group of transcription factors that belong to the superfamily of basic domain/ leucine zipper domain proteins such as cAMP-responsive element-binding protein (CREB) and activating transcription factors. All of them share high structural similarities and possess the ability to bind specifically cis-regulatory DNA sequences as homo-or heterodimers. The target motifs are palindromic DNA sequences denoted cAMP response elements (CRE, TGACGTCA) as well as the 5Ј-TGAC half-sites of this palindrome, both of which are highly conserved within numerous promoters of eukaryotic genes (6). Increased intracellular cAMP levels stimulate a multitude of kinases, e.g. calmodulin kinase IV, that subsequently phosphorylate and thereby activate CREM (7).Previous studies from our group have provided evidence that CREM␣ expression is increased in T cells from SLE patients (8). We have shown that CREM␣ binds to the Ϫ180 site of the IL-2 promoter in SLE T c...

No abstract

The transcriptional repressor cAMP response element modulator (CREM) ␣ has important roles in normal T cell physiology and contributes to aberrant T cell function in patients with systemic lupus erythematosus (SLE). Recently, we characterized a specificity protein-1-dependent promoter located upstream of the CREM gene that accounts for increased basal CREM expression in SLE T cells and reflects disease activity. Here, we identify a novel intronic CREM promoter (denoted P2) in front of the second exon of the CREM gene that harbors putative binding sites for TATA-binding proteins and the transcriptional activator AP-1. DNA binding studies, chromatin immunoprecipitation, and reporter assays confirmed the functional relevance of these sites, and T cell activation through CD3/CD28 stimulation or phorbol 12-myristate 13-acetate/ionomycin treatment enhances P2 promoter activity. Although the basal CREM levels are increased in T cells from SLE patients compared with healthy controls, there are remarkable differences in the regulation of CREM expression in response to T cell activation. Whereas T cells from healthy individuals display increased CREM expression after T cell activation, most likely through AP-1-dependent up-regulation of the P2 promoter, SLE T cells fail to further increase their basal CREM levels upon T cell activation due to a decreased content of the AP-1 family member c-Fos. Because CREM trans-represses c-fos transcription in SLE T cells, we propose an autoregulatory feedback mechanism between CREM and AP-1. Our findings extend the understanding of CREM gene regulation in the context of T cell activation and disclose another difference in the transcriptional machinery in SLE T cells.The cAMP response element modulator (CREM) 3 belongs to a superfamily of transcription factors, including cAMP response element (CRE)-binding protein, CREM, the inducible CRE repressor, and activating transcription factors, which are involved in numerous biologic processes, including immune cell homeostasis, steroid metabolism, and spermatogenesis (1-3). The cAMP-mediated signaling cascade is an evolutionarily conserved pathway that comprises stimulus-dependent cAMP production, cAMP binding to the regulatory subunits of protein kinases, nuclear translocation of the regulatory units of these kinases, and phosphorylation of the CRE-binding protein/CREM/activating transcription factors. All CREM proteins are characterized by basic domain/leucine zipper motifs; however, the multiexonic character of the CREM gene, the usage of an inducible intronic promoter or alternative initiation codons, and differential splicing mechanisms account for the presence of multiple CREM isoforms that are expressed in a cell-and development-specific fashion (4 -6). Based on the specific exon composition, CREM exerts trans-activating or trans-repressive effects on target gene expression by direct binding to CREs (TGACGTCA) or the CRE 5Ј-half-site in cisregulatory regions (6). CREs are highly conserved within numerous promoters of eukaryotic genes. T...