William H. Walker - US grants

We study the structure, function, immunology and genetics of a large class of the related proteins, bacterial pili. Pili are rod-like assemblies of identical protein molecules (pilins). Some kinds of pili have tip-attached structures consisting of differentiated pilins (adhesions and other minor proteins). The principal function of pili is to attach bacteria to specific (usually carbohydrate) receptors on host cells. The pilus site for this adhesion is located on their distal tip. Essentially all pathogenic bacteria express pili. The tissue and organ tropisms of bacterial pathogens is largely determined by the binding specificity of their pili and the distribution of pilus receptors in the host. The objectives of our research are fundamental and applied. We seek to understand the structure-function relationships in and among pili on a molecular scale. To this end we have determined the amino acid sequences of a large number of pilus proteins using both chemical and genetic techniques. We have also determined the arrangement of protein subunits in pili using X-ray diffraction and crystallographic techniques. By aligning the amino acid sequences of a number of different pilins, adhesions and other pilus proteins we have discovered a large superfamily of related pili we call ``pilus superfamily I''. This family includes pili from a number of important pathogens, for example: Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Serratia marscescens, and Hemophilus influenzae. The alignment of these sequences has revealed conserved superfamily sequences involved in common peptide folding and protein assembly patterns, conserved adhesion family sequences invol ved in binding to pilus receptors, and variable sequences involved in inter-pilus binding and/or antigenic variation as an immune evasion mechanism.

DESCRIPTION (provided by applicant): Testosterone is absolutely essential for spermatogenesis and targets Sertoli cells in the mammalian testis through the androgen receptor (AR) to produce factors required for germ cell development and survival. We now have evidence for an alternative, rapid (< 5 min) mechanism by which testosterone stimulates the phosphorylation (activation) of the Erk MAP kinase and the CREB transcription factor in primary rat Sertoli cells. This novel mechanism of testosterone action will impact a broad number of processes in Sertoli cells because MAP kinase and CREB are focal points for the control of numerous signaling pathways. Furthermore, this proposal addresses a longstanding gap in the understanding of the mechanisms by which testosterone regulates spermatogenesis as few AR-regulated genes have been identified. We will test the overall hypothesis that testosterone binding to AR activates a Src kinase and/or Ca2+-mediated signaling cascade resulting in the phosphorylation and activation of MAP kinase and CREB. Aim 1 is to test the hypothesis that AR is required to mediate the rapid phosphorylation of MAP kinase and CREB. Androgen-induced ERK and CREB phosphorylation will be assayed in normal and AR deficient rat primary Sertoli cells. AR domains required for androgen signaling will be identified. The hypothesis that populations of AR are associated with the Sertoli cell plasma membrane will be tested. Aim 2 is to test the hypothesis that androgen activates MAP kinase and CREB by a Src kinase-regulated pathway and/or via a Ca2+-mediated pathway. Androgen-induced Src kinase activity will be determined and androgen-activation of Erk and CREB wilt be assayed after addition of pharmacological and gene-based inhibitors of Src activity. Specific blockers of Ca2+ channels and Ca2+ actions will be used to test the hypothesis that Ca2+-mediated signaling pathways propagate androgen signals. Aim 3 is to test the hypothesis that androgen activates CREB and MAP kinase-regulated gene expression. Androgen regulation of MAP kinase and CREB-mediated gene expression will be tested using reporter plasmids in transient transfection assays. The regulation of specific endogenous Sertoli cell target genes via CREB will be assessed using RNAse protection assays. The information generated from this study will be directly applicable to divining the mechanisms by which testosterone supports spermatogenesis and maintains male fertility.

The long-term goal of this project is to determine the molecular and cellular mechanisms by which testosterone regulates spermatogenesis. Testosterone is essential for fertility and targets Sertoli cells in the mammalian testis through the androgen receptor (AR) to produce factors and provide an environment required for germ cell survival and development. Numerous critical Sertoli cell activities have been found to be dependent on testosterone including 1) the formation of tight junctions between Sertoli cells that form the essential blood-testis barrier, 2) the remodeling of Sertoli cell-round spermatid attachments and the retention of round spermatids, 3) the release of mature spermatozoa. Although testosterone had been known to be required for these functions, the mechanisms by which testosterone acts are not well understood. We recently characterized an alternative, rapid (<1 min) and sustained mechanism of testosterone action (the non-classical pathway) that causes the phosphorylation and activation of the Src and ERK kinases, the epidermal growth factor receptor and the CREB transcription factor. We will test the overall hypothesis that critical spermatogenesis supporting functions of testosterone in vivo are mediated via the non-classical pathway. Aim 1 is to determine whether non-classical signaling by testosterone regulates Sertoli-Sertoli and Sertoli-germ cell adhesion in culture. We will test the hypotheses that non-classical testosterone actions are required for the formation of Sertoli-Sertoli tight junctions and that non-classical signaling is required for Sertoli-germ cell adhesion. Aim 2 is to determine whether non-classical testosterone actions regulate Sertoli-germ cell interactions in seminiferous tubules. We will test the hypothesis that non-classical testosterone actions activate kinases in seminiferous tubules and cause the release of mature spermatozoa. Aim 3 is to determine whether the non-classical pathway is required to activate Src and/or ERK kinase and downstream targets required to maintain fertility. We will test the hypothesis that testosterone activates Src and ERK kinases in rat testes in vivo and that over expression of the kinases is sufficient to release mature spermatozoa. This study will identify the mechanisms by which testosterone supports spermatogenesis and assist in divining methods to regulate male fertility.

The long-term objective of this proposal is to elucidate the molecular mechanisms that regulate Sertoli cell proliferation and differentiation thereby determining the factors that limit spermatogenic capacity and male fertility. Sertoli cells are essential for the maturation and expansion of spermatozoa but the somatic cell can support only a finite number of germ cells. The size of the Sertoli cell population sets the upper limit for male fertility and is determined by the proliferative capacity of Sertoli cells prior to their terminal differentiation during puberty. The overall hypothesis to be tested is that the relative activities of Id and Ebox proteins expressed by Sertoli cells during development determine whether these somatic cells proliferate or differentiate. Aim 1 is to determine whether Id proteins regulate Sertoli cell proliferation and differentiation. Aim 2 is to determine whether up-regulation of E-box binding activity is required for Sertoli cell differentiation. Aim 3 is to identify the mechanisms employed by Id proteins and E-box transcription factors to regulate Sertoli cell development. Over expression and knockdown of Id proteins using adenoviral vectors and RNA interference (RNAi) in rat and non-human primate (rhesus monkey) Sertoli cell models will be used to determine whether Id proteins directly stimulate Sertoli cell proliferation and inhibit differentiation. E-box mRNA expression, DNA binding activity and promoter stimulating activity will be assessed using quantitative RT-PCR, DNA-protein binding assays and transient transfection assays, respectively. To determine whether Id and E-box proteins regulate genes governing Sertoli cell development, the expression of target genes will be assessed by quantitative RT-PCR after over expression and RNAi knockdown of Id and E-box proteins. Collectively, the results of these studies in both rodent and primate systems will establish the molecular mechanisms that regulate Sertoli cell proliferation and differentiation. Understanding this aspect of post-natal testicular development in both rodent and primate models will provide important insights into the pathophysiology of male infertility.