Robin M. Hobbs

Essential role of Plzf in maintenance of spermatogonial stem cells.

Little is known of the molecular mechanisms whereby spermatogonia, mitotic germ cells of the testis, self-renew and differentiate into sperm. 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/Wv 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 spermatogonia-specific transcription factor in the testis that is required to regulate self-renewal and maintenance of the stem cell pool.

The BTB–zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions

Invariant natural killer T cells (iNKT cells) have an innate immunity–like rapidity of response and the ability to modulate the effector functions of other cells. We show here that iNKT cells specifically expressed the BTB–zinc finger transcriptional regulator PLZF. In the absence of PLZF, iNKT cells developed, but they lacked many features of innate T cells. PLZF-deficient iNKT cells accumulated in lymph nodes rather than in the liver, did not express NK markers and did not have the characteristic activated phenotype. PLZF-deficient iNKT cells failed to secrete large amounts of interleukin 4 and interferon-γ after activation; however, some cells produced either interleukin 4 or interferon-γ but not both. PLZF, therefore, is an iNKT cell–specific transcription factor that is necessary for full functionality.

Identification of the miR-106b∼25 microRNA cluster as a proto-oncogenic PTEN-targeting intron that cooperates with its host gene MCM7 in transformation

A microRNA network regulates the tumor suppressor PTEN in prostate cancer. A Malignant Combination The abundance of microRNAs (miRNAs), tiny non–protein-coding RNAs that act as posttranscriptional regulators of gene expression, is frequently altered in cancer; indeed, various miRNAs are thought to act as oncogenes or tumor suppressors. Poliseno et al. investigated the possible role of miRNA regulation of the tumor suppressor PTEN in prostate cancer. They identified miRNAs from several families that targeted the gene encoding PTEN, thereby decreasing PTEN abundance, and showed that the abundance of some of these miRNAs was increased in human prostate cancer. Intriguingly, three PTEN-targeting miRNAs located within an intron of the gene encoding the DNA helicase minichromosome maintenance protein 7 (MCM7), which shows increased abundance in various human cancers, cooperated with MCM7 to transform fibroblasts in vitro and to initiate tumors when overexpressed in the prostates of transgenic mice. Thus, the MCM7 gene locus appears to encode multiple oncogenic elements that cooperate to promote prostate cancer development. 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 proto-oncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.

A Novel Signal Transduction Cascade Involving Direct Physical Interaction of the Renin/Prorenin Receptor With the Transcription Factor Promyelocytic Zinc Finger Protein

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 p85α subunit of the phosphatidylinositol-3 kinase (PI3K-p85α). 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-p85α. 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.

Role of the proto-oncogene Pokemon in cellular transformation and ARF repression

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. Pokemons critical role in cellular transformation makes it an attractive target for therapeutic intervention.

TCR-inducible PLZF transcription factor required for innate phenotype of a subset of γδ T cells with restricted TCR diversity

Some γδ and αβ T lymphocytes exhibit an “innate” phenotype associated with rapid cytokine responses. The PLZF transcription factor is essential for the innate phenotype of NKT cells. This report shows that PLZF is likewise responsible for the innate, NKT-like phenotype of Vγ1+Vδ6.3/Vδ6.4+ cells. TCR cross-linking induced PLZF expression in all polyclonal immature γδ thymocytes, suggesting that agonist selection might be required for PLZF induction. Transgenic expression of Vγ1Vδ6.4 TCR was sufficient to support the development of large numbers of PLZF+ T cells, further supporting the importance of the TCR for PLZF induction. Interestingly, expression of this TCR transgene led to the development of spontaneous dermatitis.

Characterization, Cryopreservation, and Ablation of Spermatogonial Stem Cells in Adult Rhesus Macaques

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare Asingle spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant Adark and Apale spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high‐dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRα1), and cells expressing these markers were depleted following high‐dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high‐dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell‐depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.

Plzf Regulates Germline Progenitor Self-Renewal by Opposing mTORC1

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.

A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis

In normal epidermis, beta1 integrin expression is confined to the basal layer, whereas in hyperproliferative epidermis, integrins are also expressed in the suprabasal layers. Transgenic mice in which integrins are expressed suprabasally via the involucrin promoter have a sporadic psoriatic phenotype; however, the mechanism by which integrins contribute to the pathogenesis of psoriasis is unknown. We observed activation of mitogen-activated protein kinase (MAPK) in basal and suprabasal keratinocytes of human and transgenic mouse psoriatic lesions and healing mouse skin wounds, correlating in each case with suprabasal integrin expression. Phenotypically normal human and transgenic mouse epidermis did not contain activated MAPK. Transgene-positive keratinocytes produced more IL-1alpha than controls did, and keratinocyte MAPK could be activated by ligation of suprabasal integrins or treatment with IL-1alpha. Constitutive activation of MAPK increased the growth rate of human keratinocytes and delayed the onset of terminal differentiation, recreating many of the histological features of psoriatic epidermis. We propose that activation of MAPK by integrins, either directly or through increased IL-1alpha production, is responsible for epidermal hyperproliferation in psoriasis and wound healing, and that the sporadic phenotype of the transgenic mice may reflect the complex mechanisms by which IL-1 release and responsiveness are controlled in skin.

Repression of kit Expression by Plzf in Germ Cells

ABSTRACT Male mice lacking expression of Plzf, a DNA sequence-specific transcriptional repressor, show progressive germ cell depletion due to exhaustion of the spermatogonial stem cell population. This is likely due to the deregulated expression of genes controlling the switch between spermatogonial self-renewal and differentiation. Here we show that Plzf directly represses the transcription of kit, a hallmark of spermatogonial differentiation. Plzf represses both endogenous kit expression and expression of a reporter gene under the control of the kit promoter region. A discrete sequence of the kit promoter, required for Plzf-mediated kit transcriptional repression, is bound by Plzf both in vivo and in vitro. A 3-bp mutation in this Plzf binding site abolishes the responsiveness of the kit promoter to Plzf repression. A significant increase in kit expression is also found in the undifferentiated spermatogonia isolated from Plzf−/− mice. Thus, we suggest that one mechanism by which Plzf maintains the pool of spermatogonial stem cells is through a direct repression of kit expression.