Single-cell analyses unravel cell type-specific responses to a vitamin D analog in prostatic precancerous lesions.

Epidemiological data have linked vitamin D deficiency to the onset and severity of various cancers, including prostate cancer, and although in vitro studies have demonstrated anticancer activities for vitamin D, clinical trials provided conflicting results. To determine the impact of vitamin D signaling on prostatic precancerous lesions, we treated genetically engineered Pten(i)pe-/- mice harboring prostatic intraepithelial neoplasia (PIN) with Gemini-72, a vitamin D analog with reported anticancer activities. We show that this analog induces apoptosis in senescent PINs, normalizes extracellular matrix remodeling by stromal fibroblasts, and reduces the prostatic infiltration of immunosuppressive myeloid-derived suppressor cells. Moreover, single-cell RNA-sequencing analysis demonstrates that while a subset of luminal cells expressing Krt8, Krt4, and Tacstd2 (termed luminal-C cells) is lost by such a treatment, antiapoptotic pathways are induced in persistent luminal-C cells. Therefore, our findings delineate the distinct responses of PINs and the microenvironment to Gemini-72, and shed light on mechanisms that limit treatment's efficacy.

PTEN deletion in luminal cells of mature prostate induces replication stress and senescence in vivo.

Genetic ablation of the tumor suppressor PTEN in prostatic epithelial cells (PECs) induces cell senescence. However, unlike oncogene-induced senescence, no hyperproliferation phase and no signs of DNA damage response (DDR) were observed in PTEN-deficient PECs; PTEN loss-induced senescence (PICS) was reported to be a novel type of cellular senescence. Our study reveals that PTEN ablation in prostatic luminal epithelial cells of adult mice stimulates PEC proliferation, followed by a progressive growth arrest with characteristics of cell senescence. Importantly, we also show that proliferating PTEN-deficient PECs undergo replication stress and mount a DDR leading to p53 stabilization, which is however delayed by Mdm2-mediated p53 down-regulation. Thus, even though PTEN-deficiency induces cellular senescence that restrains tumor progression, as it involves replication stress, strategies promoting PTEN loss-induced senescence are at risk for cancer prevention and therapy.

The transcriptional coregulator PGC-1ß controls mitochondrial function and anti-oxidant defence in skeletal muscles.

The transcriptional coregulators PGC-1α and PGC-1ß modulate the expression of numerous partially overlapping genes involved in mitochondrial biogenesis and energetic metabolism. The physiological role of PGC-1ß is poorly understood in skeletal muscle, a tissue of high mitochondrial content to produce ATP levels required for sustained contractions. Here we determine the physiological role of PGC-1ß in skeletal muscle using mice, in which PGC-1ß is selectively ablated in skeletal myofibres at adulthood (PGC-1ß((i)skm-/-) mice). We show that myofibre myosin heavy chain composition and mitochondrial number, muscle strength and glucose homeostasis are unaffected in PGC-1ß((i)skm-/-) mice. However, decreased expression of genes controlling mitochondrial protein import, translational machinery and energy metabolism in PGC-1ß((i)skm-/-) muscles leads to mitochondrial structural and functional abnormalities, impaired muscle oxidative capacity and reduced exercise performance. Moreover, enhanced free-radical leak and reduced expression of the mitochondrial anti-oxidant enzyme Sod2 increase muscle oxidative stress. PGC-1ß is therefore instrumental for skeletal muscles to cope with high energetic demands.

Temporally controlled targeted somatic mutagenesis in mouse eye pigment epithelium.

To generate temporally controlled site-specific somatic mutations in the mouse eye pigment epithelium, we generated a TRP1-Cre-ER(T2) transgenic mouse line that expresses the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the tyrosinase-related protein 1 (TRP1) promoter. Cre-ER(T2) transcripts were readily detected in the retinal pigment epithelium (RPE), and tamoxifen treatment of adult TRP1-Cre-ER(T2) transgenic mice induced efficient excision of floxed DNA in patches of RPE cells, in numerous epithelial cells of the iris and ciliary body, and in very few cells of the neural retina. Importantly, no excision was detected in any cells in the absence of tamoxifen treatment. Thus, the TRP1-Cre-ER(T2) mouse line provides a powerful tool to study in vivo gene functions in the mouse eye pigment epithelium.

Ctip2/Bcl11b controls ameloblast formation during mammalian odontogenesis.

The transcription factor Ctip2/Bcl11b plays essential roles in developmental processes of the immune and central nervous systems and skin. Here we show that Ctip2 also plays a key role in tooth development. Ctip2 is highly expressed in the ectodermal components of the developing tooth, including inner and outer enamel epithelia, stellate reticulum, stratum intermedium, and the ameloblast cell lineage. In Ctip2(-/-) mice, tooth morphogenesis appeared to proceed normally through the cap stage but developed multiple defects at the bell stage. Mutant incisors and molars were reduced in size and exhibited hypoplasticity of the stellate reticulum. An ameloblast-like cell population developed ectopically on the lingual aspect of mutant lower incisors, and the morphology, polarization, and adhesion properties of ameloblasts on the labial side of these teeth were severely disrupted. Perturbations of gene expression were also observed in the mandible of Ctip2(-/-) mice: expression of the ameloblast markers amelogenin, ameloblastin, and enamelin was down-regulated, as was expression of Msx2 and epiprofin, transcription factors implicated in the tooth development and ameloblast differentiation. These results suggest that Ctip2 functions as a critical regulator of epithelial cell fate and differentiation during tooth morphogenesis.

Dual role of COUP-TF-interacting protein 2 in epidermal homeostasis and permeability barrier formation.

COUP-TF-interacting protein 2 (CTIP2; also known as Bcl11b) is a transcription factor that plays key roles in the development of the central nervous and immune systems. CTIP2 is also highly expressed in the developing epidermis, and at lower levels in the dermis and in adult skin. Analyses of mice harboring a germline deletion of CTIP2 revealed that the protein plays critical roles in skin during development, particularly in keratinocyte proliferation and late differentiation events, as well as in the development of the epidermal permeability barrier. At the core of all of these actions is a relatively large network of genes, described herein, that is regulated directly or indirectly by CTIP2. The analysis of conditionally null mice, in which expression of CTIP2 was ablated specifically in epidermal keratinocytes, suggests that CTIP2 functions in both cell and non-cell autonomous contexts to exert regulatory influence over multiple phases of skin development, including barrier establishment. Considered together, our results suggest that CTIP2 functions as a top-level regulator of skin morphogenesis.

Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse.

To generate temporally-controlled targeted somatic mutations selectively and efficiently in smooth muscles, we have established a transgenic SMA-Cre-ER(T2) mouse line in which the expression of the Tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the mouse smooth muscle alpha actin (SMA) gene, contained in a Bacterial artificial chromosome (Bac). In this transgenic mouse line, Cre-ER(T2)-mediated recombination of LoxP-flanked target DNA is strictly Tamoxifen-dependent, and efficient in both vascular and visceral smooth muscle cells. Moreover, with the exception of few cardiomyocytes, LoxP-flanked DNA excision is restricted to smooth muscle cells. Thus, SMA-Cre-ER(T2) mice should be of great value to analyze gene function in smooth muscles, and to establish new animal models of human smooth muscle disorders.

PGC1alpha expression is controlled in skeletal muscles by PPARbeta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes.

Mice in which peroxisome proliferator-activated receptor beta (PPARbeta) is selectively ablated in skeletal muscle myocytes were generated to elucidate the role played by PPARbeta signaling in these myocytes. These somatic mutant mice exhibited a muscle fiber-type switching toward lower oxidative capacity that preceded the development of obesity and diabetes, thus demonstrating that PPARbeta is instrumental in myocytes to the maintenance of oxidative fibers and that fiber-type switching is likely to be the cause and not the consequence of these metabolic disorders. We also show that PPARbeta stimulates in myocytes the expression of PGC1alpha, a coactivator of various transcription factors, known to play an important role in slow muscle fiber formation. Moreover, as the PGC1alpha promoter contains a PPAR response element, the effect of PPARbeta on the formation and/or maintenance of slow muscle fibers can be ascribed, at least in part, to a stimulation of PGC1alpha expression at the transcriptional level.

Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation.

Animal SWI2/SNF2 protein complexes containing either the brahma (BRM) or brahma-related gene 1 (BRG1) ATPase are involved in nucleosome remodelling and may control the accessibility of sequence-specific transcription factors to DNA. In vitro studies have indicated that BRM and BRG1 could regulate the expression of distinct sets of genes. However, as mice lacking BRM are viable and fertile, BRG1 might efficiently compensate for BRM loss. By contrast, as Brg1-null fibroblasts are viable but Brg1-null embryos die during the peri-implantation stage, BRG1 might exert cell-specific functions. To further investigate the in vivo role of BRG1, we selectively ablated Brg1 in keratinocytes of the forming mouse epidermis. We show that BRG1 is selectively required for epithelial-mesenchymal interactions in limb patterning, and during keratinocyte terminal differentiation, in which BRM can partially substitute for BRG1. By contrast, neither BRM nor BRG1 are essential for the proliferation and early differentiation of keratinocytes, which may require other ATP-dependent nucleosome-remodelling complexes. Finally, we demonstrate that cell-specific targeted somatic mutations can be created at various times during the development of mouse embryos cell-specifically expressing the tamoxifen-activatable Cre-ER(T2) recombinase.

Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse.

The peroxisome proliferator-activated receptor gamma (PPARgamma) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARgamma functions in mature adipocytes is precluded by lethality of PPARgamma(-/-) fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARgamma in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER(T2) recombination system. We show that mature PPARgamma-null white and brown adipocytes die within a few days and are replaced by newly formed PPARgamma-positive adipocytes, demonstrating that PPARgamma is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARgamma antagonists could be used to acutely reduce obesity.