Little is known of the molecular mechanisms whereby spermatogonia, mitotic germ cells of the testis, self-renew and differentiate into sperm 1,2 . Here we show that Zfp145, encoding the transcriptional repressor Plzf, has a crucial role in spermatogenesis. Zfp145 expression was restricted to gonocytes and undifferentiated spermatogonia and was absent in tubules of W/W v mutants that lack these cells. Mice lacking Zfp145 underwent a progressive loss of spermatogonia with age, associated with increases in apoptosis and subsequent loss of tubule structure but without overt differentiation defects or loss of the supporting Sertoli cells. Spermatogonial transplantation experiments revealed a depletion of spermatogonial stem cells in the adult. Microarray analysis of isolated spermatogonia from Zfp145-null mice before testis degeneration showed alterations in the expression profile of genes associated with spermatogenesis. These results identify Plzf as a spermatogoniaspecific transcription factor in the testis that is required to regulate self-renewal and maintenance of the stem cell pool.

Invariant Natural Killer T cell (iNK Tcell) have an innate immunity-like rapidity of response and the capacity to modulate effector functions of other cells. We show that the BTB-ZF transcriptional regulator, PLZF, is specifically expressed in iNKT cells. iNKT cells develop in the absence of PLZF, but lack many innate T cell features. PLZF deficient iNKT cells accumulate in the lymph nodes rather than in the liver and do not have an activated phenotype or express NK markers. PLZF deficient iNKT cells do not secrete high levels of IL-4 and IFNγ upon activation, however some cells produce either IL-4 or IFNγ, but not both. PLZF, therefore, is an iNKT cell specific transcription factor that is necessary for full functionality.Nearly all hematopoietic cells mature in the bone marrow. In contrast, multipotent progenitor T cells leave the bone marrow and home to the thymus, where signals from stromal cells are required for commitment to the T lineage 1 . Once directed into the T lineage, the cells undergo a rigorous selection process that eliminates more than 95% of the candidate T cells. Full maturation requires the expression of a T cell receptor (TCR) that binds self-peptide:self-MHC complexes with sufficient avidity. At some point during development, T cells are directed into one of several distinct T cell lineages such as CD4 single positive "helper" cells, CD8 single positive "killer" cells or CD4 + CD25 + regulatory cells. Commitment to these various lineages defines the specialized functions of the cell, which is critical since each cell type plays an essential and distinct role for host defense. The genes responsible for directing multipotent T cell progenitors into the various lineages are largely unknown 2 .Correspondence and requests for materials should be addressed to D.B.S (Email: santangd@mskcc.org). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAmong the various lineages of T cells, invariant Natural Killer T cells (iNKT cells) have several unique phenotypic traits such as the expression of receptors typically associated with Natural Killer cells (NK cells), the constitutive expression of activation markers and extremely restricted TCR diversity 3 . iNKT cells express an identical TCRα chain and most use a TCRβ chain that utilizes the Vβ8.2 gene segment. This TCR confers specificity to the non-MHC encoded self-molecule, CD1d, which binds and presents glycolipids rather than the typical peptide cargo presented by conventional MHC molecules.iNKT cells are also functionally distinct. Of particular interest is their ability to secrete large quantities of a variety of cytokines only minutes after activation via the TCR 3 . The rapid response of these cells, the conserved nature of the TCR and their indirect ability to modulate the function of many different cell types of the immune system has led to the appreciation that iNKT cells lay at a functional cusp between the innate and adaptive immune systems 4 . The broad range of cytokines released by iNKT cells results in th...

Aberrant transcriptional repression through chromatin remodelling and histone deacetylation has been postulated to represent a driving force underlying tumorigenesis because histone deacetylase inhibitors have been found to be effective in cancer treatment. However, the molecular mechanisms by which transcriptional derepression would be linked to tumour suppression are poorly understood. Here we identify the transcriptional repressor Pokemon (encoded by the Zbtb7 gene) as a critical factor in oncogenesis. Mouse embryonic fibroblasts lacking Zbtb7 are completely refractory to oncogene-mediated cellular transformation. Conversely, Pokemon overexpression leads to overt oncogenic transformation both in vitro and in vivo in transgenic mice. Pokemon can specifically repress the transcription of the tumour suppressor gene ARF through direct binding. We find that Pokemon is aberrantly overexpressed in human cancers and that its expression levels predict biological behaviour and clinical outcome. Pokemon's critical role in cellular transformation makes it an attractive target for therapeutic intervention.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol-3-kinase-Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b∼25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b∼25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a protooncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.

Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

SUMMARY Hyperactivity of mTORC1, a key mediator of cell growth, leads to stem cell depletion although the underlying mechanisms are poorly defined. Using spermatogonial progenitor cells (SPCs) as a model system, we show that mTORC1 impairs stem cell maintenance by a negative feedback from mTORC1 to receptors required to transduce niche-derived signals. We find that SPCs lacking Plzf, a transcription factor essential for SPC maintenance, have enhanced mTORC1 activity. Aberrant mTORC1 activation in Plzf −/− SPCs inhibits their response to GDNF, a growth factor critical for SPC self-renewal, via negative feedback at the level of the GDNF receptor. Plzf opposes mTORC1 activity by inducing expression of the mTORC1 inhibitor Redd1. Thus, we identify the mTORC1-Plzf functional interaction as a critical rheostat for maintenance of the spermatogonial pool, and propose a model whereby negative feedback from mTORC1 to the GDNF receptor balances SPC growth with self-renewal.

Abstract-A human renin/prorenin receptor (RER) has recently been cloned. To gain insight into the molecular function of the RER, we studied its signal transduction mechanisms. Initially, we found a ubiquitous and intracellular expression pattern of the human RER. Consistently, we observed several transcriptional start sites and a high promoter activity of the human RER. We could identify the transcription factor promyelocytic zinc finger (PLZF) protein as a direct protein interaction partner of the C-terminal domain of the RER by yeast 2-hybrid screening and coimmunoprecipitation. Coimmunoprecipitation experiments also indicated homodimerization of the RER. On activation of the RER by renin, PLZF is translocated into the nucleus and represses transcription of the RER itself, thereby creating a very short negative feedback loop, but activates transcription of the p85␣ subunit of the phosphatidylinositol-3 kinase (PI3K-p85␣). Small interfering RNA against the RER abolished these effects. A PLZF cis-element in the RER promoter was identified by site-directed mutagenesis and electrophoretic mobility-shift assay. Renin stimulation caused a 6-fold recruitment of PLZF to this promoter region as shown by chromatin immunoprecipitation. Moreover, renin stimulation of rat H9c2 cardiomyoblasts induced an increase of cell number and a decrease of apoptosis. These effects were partly abolished by PI3K inhibition and completely abrogated by small interfering RNA against PLZF. Finally, experiments in PLZF knockout mice confirmed the role of PLZF as an upstream regulator of RER and PI3K-p85␣. Our data demonstrate the existence of a novel signal transduction pathway involving the ligand renin, RER, and the transcription factor PLZF, which is of physiological and putative pathophysiological relevance. Key Words: renin receptor Ⅲ PLZF Ⅲ ChIP Ⅲ signal transduction R enin and prorenin are classically thought of as (pro)enzymes of the renin/angiotensin system (RAS), but recent evidence suggests that they also act as hormones because of their ability to bind cellular targets. 1 In 2002 a human renin/prorenin receptor (RER) has been cloned, which consists of 350 amino acids with a single transmembrane domain and specifically binds prorenin and renin. Interestingly, this receptor exerts a dual molecular function 2,3 (1) Binding of renin to its receptor increases the catalytic activity of renin approximately 4-to 5-fold. Furthermore, prorenin, which does not exhibit significant ability to generate angiotensin I in solution, gains enzymatic activity comparable to renin by binding to the RER, ie, the receptor is able to unmask the catalytic activity of prorenin. (2) The RER is also able to induce a signal transduction cascade on ligand binding. Binding of renin and also prorenin causes phosphorylation of the receptor and activation of the mitogen-activated protein kinases ERK 1 and 2 (extracellular signal-regulated kinases 1 and 2), whereas intracellular calcium or cAMP levels are not altered. Remarkably, even deglycosylated renin is able t...

SUMMARY Decremental loss of PTEN results in cancer susceptibility and tumor progression. In turn this raises the possibility that PTEN elevation might be an attractive option for cancer prevention and therapy. We have generated several transgenic mouse lines with variably elevated PTEN expression levels, taking advantage of BAC (Bacterial Artificial Chromosome)-mediated transgenesis. Super-PTEN mutants are viable and show reduced body size due to decreased cell number, with no effect on cell size. Unexpectedly, PTEN elevation at the organism level results in healthy metabolism characterized by increased energy expenditure and reduced body fat accumulation. Cells derived from these mice show reduced glucose and glutamine uptake, increased mitochondrial oxidative phosphorylation, and are resistant to oncogenic transformation. Mechanistically we find that PTEN elevation orchestrates this metabolic switch by regulating PI3K-dependent and independent pathways, and negatively impacts two of the most pronounced metabolic features of tumor cells: glutaminolysis and the Warburg effect.