SUMO modification of nuclear receptors, including the constitutively active receptor steroidogenic factor 1 (SF-1; NR5A1), is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines (Lys119 and Lys194) that reside adjacent to the DNA-binding domain (DBD) and ligand-binding domain (LBD), respectively. Surprisingly, while previous loss-of-function studies predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein was either unchanged or modestly impaired. Sumoylation at Lys194 also modestly reduced Ser203 phosphorylation. In contrast to these findings, sumoylation of the DBD at Lys119 resulted in a marked and selective loss of DNA binding to noncanonical SF-1 targets, such as inhibin␣; this binding deficit was extended to all sites when the sumoylated human mutant (R92Q) protein, which exhibits lower activity, was used. Consistent with this result, the K119R mutant, compared to wild-type SF-1, was selectively recruited to a "SUMO-sensitive" site in the endogenous inhibin␣ promoter, leading to increased transcription. DNA binding and sumoylation of Lys119 appeared to be mutually exclusive, suggesting that once SF-1 is bound to DNA, sumoylation may be less important in regulating SF-1 activity. We propose that sumoylation of nuclear receptors imposes an active posttranslational mark that dampens recognition of SUMO-sensitive target genes to restrain their expression.Posttranslational modification with ubiquitin-like proteins has emerged as an important regulatory mechanism in many aspects of cellular function (29). Indeed, the small ubiquitinlike modifier (SUMO) conjugate modifies many transcription factors and results in marked transcriptional repression (27). Sumoylation occurs on lysines within consensus KxE sites through an enzymatic mechanism analogous to ubiquitination (6). While the requirement for an obligate SUMO E3 ligase is still debated, several proteins exhibit SUMO E3 ligase activity in cells, with the largest group belonging to the protein inhibitors of activated STATs (PIAS) family (47). Sumoylation is easily reversible by the action of SUMO isopeptidases (SUSPs or SENPs), with seven members identified in humans thus far (40).While recent structural studies have helped to elucidate the enzymatic details of SUMO conjugation and substrate recognition (44, 45), the mechanisms underlying transcriptional repression by sumoylation are less clear. Identification of a SUMO-interacting motif found in the PML protein and transcriptional repressors such as Daxx has helped to establish how the SUMO conjugate might function as a passive molecular mark to attenuate gene expression (28,39). However, it is also plausible that the SUMO conjugate functions as an active mark to modify either protein-protein or protein-DNA interactions. Evidence for this latter hypothesis remains controversial. For instance, sumoylation of thymine-...
The Sertoli cell intermediate filament cytoskeleton is composed of the type III family member vimentin. The distribution of Sertoli cell vimentin varies with the stage of spermatogenesis, with shortening of the filaments at stages VII-VIII, the stages of spermiation. Experimental reduction in intratesticular testosterone (T) concentration also results in the sloughing of advanced spermatids from the Sertoli cells, as well as in the apoptotic death of spermatocytes. We hypothesized that alteration of the distribution of Sertoli cell vimentin might play a role in the loss of germ cells that occurs in response to reduced intratesticular T. To test this hypothesis, intratesticular T was reduced by implanting LH-suppressive SILASTIC brand capsules containing T and estradiol into adult rats for 8 wk. Immunohistochemical analyses revealed that, in response to the implants, the vimentin cytoskeleton collapsed around the Sertoli cell nuclei at all stages of the cycle, losing the extensive branching and structure normally seen at most stages of the cycle. Western blots of isolated Sertoli cells revealed that protein levels did not differ significantly between control and T- and estradiol-treated rats. However, Sertoli cell fractions containing the vimentin monomer revealed that vimentin was cleaved into four to five fragments in Sertoli cells in response to the implants, suggestive of proteolysis. These results indicate that, in response to reduced intratesticular T, the vimentin cytoskeleton of the Sertoli cell collapses to a perinuclear localization, and suggest that this collapse is associated with, and perhaps caused by, the degradation of the vimentin monomer rather than by loss of its expression.
Successful spermatogenesis requires that germ cells remain in physical contact with Sertoli cells until spermiation. Previous studies have shown that the Bcl2-modifying factor (BMF) is a proapoptotic protein found in many epithelial cells which, when phosphorylated by the active form of mitogen-activated protein kinase 8 (p-MAPK8), initiates apoptosis in response to loss of adhesion of the cells to their basal lamina. Based on this, we hypothesized that p-MAPK8 and BMF may play important roles in the apoptotic death of testicular germ cells in response to their detachment from Sertoli cells. Immunohistochemical analysis of the normal rat testis revealed p-MAPK8 expression in spermatocytes and elongated spermatids but not in round spermatids. This localization was opposite to that of BMF, which is expressed in round spermatids but not in spermatocytes or elongated spermatids.When freshly isolated germ cells were cultured in the absence of Sertoli cells, a condition in which there was widespread germ cell apoptosis, an increase in p-MAPK8 relative to overall MAPK8 protein, was seen by Western blot analysis. Additionally, immunocytochemical analysis showed an increase in immunoreactive p-MAPK8 in round spermatids and spermatocytes in association with BMF expression. From these correlative data, we propose that the activation of MAPK8 and redistribution of BMF may be integrally involved in the mechanism by which specific germ cells undergo programmed cell death in response to their detachment from Sertoli cells.
The Bcl2 modifying factor (Bmf) is a pro-apoptotic member of the Bcl2 family of apoptosis-related proteins that has been shown to initiate apoptosis in response to the loss of attachment of cells from their basal lamina (anoikis). Experimental reduction in intratesticular testosterone concentration brings about the death of spermatids as a consequence of their sloughing from Sertoli cells. Given the role of Bmf in anoikis in other systems, we hypothesized that Bmf would be expressed in germ cells and that its expression and normal distribution might be altered under conditions that induce widespread germ cell loss. To test these hypotheses, we demonstrated that Bmf indeed is expressed in the testis and cloned the full-length rat Bmf cDNA. Immunohistochemistry revealed that Bmf is present in the subacrosomal space of postmeiotic spermatids from step 4 to 16 of spermiogenesis. To test the hypothesis that Bmf expression and distribution are altered by conditions that elicit anoikis, intratesticular testosterone was reduced by implanting Silastic capsules containing testosterone and estradiol into adult rats for 8 weeks. As hypothesized, this resulted in a significant change in Bmf distribution relative to untreated animals. In particular, Bmf exhibited a loss of its normal subacrosomal distribution, becoming redistributed throughout the cytoplasm and nucleus, and appeared in cells in which it is not normally expressed (e.g., pachytene spermatocytes). Additionally, Bmf mRNA expression increased in response to lowered testosterone. These results suggest that Bmf may well be involved in germ cell apoptosis and/or anoikis in response to decreased intratesticular testosterone concentration.
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