Tissue-specific progesterone receptor-chromatin binding and the regulation of progesterone-dependent gene expression.

Progesterone receptor (PGR) co-ordinately regulates ovulation, fertilisation and embryo implantation through tissue-specific actions, but the mechanisms for divergent PGR action are poorly understood. Here we characterised PGR activity in mouse granulosa cells using combined ChIP-seq for PGR and H3K27ac and gene expression microarray. Comparison of granulosa, uterus and oviduct PGR-dependent genes showed almost complete tissue specificity in PGR target gene profiles. In granulosa cells 82% of identified PGR-regulated genes bound PGR within 3 kb of the gene and PGR binding sites were highly enriched in proximal promoter regions in close proximity to H3K27ac-modified active chromatin. Motif analysis showed highly enriched PGR binding to the PGR response element (GnACAnnnTGTnC), but PGR also interacted significantly with other transcription factor binding motifs. In uterus PGR showed far more tendency to bind intergenic chromatin regions and low evidence of interaction with other transcription factors. This is the first genome-wide description of PGR action in granulosa cells and systematic comparison of diverse PGR action in different reproductive tissues. It clarifies finely-tuned contextual PGR-chromatin interactions with implications for more targeted reproductive medicine.

Uterine RAC1 via Pak1-ERM signaling directs normal luminal epithelial integrity conducive to on-time embryo implantation in mice.

Successful embryo implantation requires functional luminal epithelia to establish uterine receptivity and blastocyst-uterine adhesion. During the configuration of uterine receptivity from prereceptive phase, the luminal epithelium undergoes dynamic membrane reorganization and depolarization. This timely regulated epithelial membrane maturation and precisely maintained epithelial integrity are critical for embryo implantation in both humans and mice. However, it remained largely unexplored with respect to potential signaling cascades governing this functional epithelial transformation prior to implantation. Using multiple genetic and cellular approaches combined with uterine conditional Rac1 deletion mouse model, we demonstrated herein that Rac1, a small GTPase, is spatiotemporally expressed in the periimplantation uterus, and uterine depletion of Rac1 induces premature decrease of epithelial apical-basal polarity and defective junction remodeling, leading to disrupted uterine receptivity and implantation failure. Further investigations identified Pak1-ERM as a downstream signaling cascade upon Rac1 activation in the luminal epithelium necessary for uterine receptivity. In addition, we also demonstrated that Rac1 via P38 MAPK signaling ensures timely epithelial apoptotic death at postimplantation. Besides uncovering a potentially important molecule machinery governing uterine luminal integrity for embryo implantation, our finding has high clinical relevance, because Rac1 is essential for normal endometrial functions in women.

Targeting the glucose-regulated protein-78 abrogates Pten-null driven AKT activation and endometrioid tumorigenesis.

Rates of the most common gynecologic cancer, endometrioid adenocarcinoma (EAC), continue to rise, mirroring the global epidemic of obesity, a well-known EAC risk factor. Thus, identifying novel molecular targets to prevent and/or mitigate EAC is imperative. The prevalent Type 1 EAC commonly harbors loss of the tumor suppressor, Pten, leading to AKT activation. The major endoplasmic reticulum (ER) chaperone, GRP78, is a potent pro-survival protein to maintain ER homeostasis, and as a cell surface protein, is known to regulate the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. To determine whether targeting GRP78 could suppress EAC development, we created a conditional knockout mouse model using progesterone receptor-Cre-recombinase to achieve Pten and Grp78 (cPten(f/f)Grp78(f/f)) deletion in the endometrial epithelium. Mice with a single Pten (cPten(f/f)) deletion developed well-differentiated EAC by 4 weeks. In contrast, no cPten(f/f)Grp78(f/f) mice developed EAC, even after more than 8 months of observation. Histologic examination of uteri from cPten(f/f)Grp78(f/f) mice also revealed no complex atypical hyperplasia, a well-established EAC precursor. These histologic observations among the cPten(f/f)Grp78(f/f) murine uteri also corresponded to abrogation of AKT activation within the endometrium. We further observed that GRP78 co-localized with activated AKT on the surface of EAC, thus providing an opportunity for therapeutic targeting. Consistent with previous findings that cell surface GRP78 is an upstream regulator of PI3K/AKT signaling, we show here that in vivo short-term systemic treatment with a highly specific monoclonal antibody against GRP78 suppressed AKT activation and increased apoptosis in the cPten(f/f) tumors. Collectively, these findings present GRP78-targeting therapy as an efficacious therapeutic option for EAC.

Loss of Cdh1 and Trp53 in the uterus induces chronic inflammation with modification of tumor microenvironment.

Type II endometrial carcinomas (ECs) are estrogen independent, poorly differentiated tumors that behave in an aggressive manner. As TP53 mutation and CDH1 inactivation occur in 80% of human endometrial type II carcinomas, we hypothesized that mouse uteri lacking both Trp53 and Cdh1 would exhibit a phenotype indicative of neoplastic transformation. Mice with conditional ablation of Cdh1 and Trp53 (Cdh1(d/d)Trp53(d/d)) clearly demonstrate architectural features characteristic of type II ECs, including focal areas of papillary differentiation, protruding cytoplasm into the lumen (hobnailing) and severe nuclear atypia at 6 months of age. Further, Cdh1(d/d)Trp53(d/d) tumors in 12-month-old mice were highly aggressive, and metastasized to nearby and distant organs within the peritoneal cavity, such as abdominal lymph nodes, mesentery and peri-intestinal adipose tissues, demonstrating that tumorigenesis in this model proceeds through the universally recognized morphological intermediates associated with type II endometrial neoplasia. We also observed abundant cell proliferation and complex angiogenesis in the uteri of Cdh1(d/d)Trp53(d/d) mice. Our microarray analysis found that most of the genes differentially regulated in the uteri of Cdh1(d/d)Trp53(d/d) mice were involved in inflammatory responses. CD163 and Arg1, markers for tumor-associated macrophages, were also detected and increased in the uteri of Cdh1(d/d)Trp53(d/d) mice, suggesting that an inflammatory tumor microenvironment with immune cell recruitment is augmenting tumor development in Cdh1(d/d)Trp53(d/d) uteri. Further, inflammatory mediators secreted from CDH1-negative, TP53 mutant endometrial cancer cells induced normal macrophages to express inflammatory-related genes through activation of nuclear factor-κB signaling. These results indicate that absence of CDH1 and TP53 in endometrial cells initiates chronic inflammation, promotes tumor microenvironment development following the recruitment of macrophages and promotes aggressive ECs.

Role of nuclear receptors in blastocyst implantation.

The regulation of blastocyst implantation in the uterus is orchestrated by the ovarian hormones estrogen and progesterone. These hormones act via their nuclear receptors to direct the transcriptional activity of the endometrial compartments and create a defined period in which the uterus is permissive to embryo implantation termed the "window of receptivity". Additional members of the nuclear receptor family have also been described to have a potential role in endometrial function. Much of what we know about the function of these nuclear receptors during implantation we have learned from the use of mouse models. Transgenic murine models with targeted gene ablation have allowed us to identify a complex network of paracrine signaling between the endometrial epithelium and stroma. While some of the critical molecules have been identified, the mechanism underlying the intricate communication between endometrial compartments during the implantation window has not been fully elucidated. Defining this mechanism will help identify markers of a receptive uterine environment, ultimately providing a useful tool to help improve the fertility outlook for reproductively challenged couples. The aim of this review is to outline our current understanding of how nuclear receptors and their effector molecules regulate blastocyst implantation in the endometrium.

beta-catenin mediates glandular formation and dysregulation of beta-catenin induces hyperplasia formation in the murine uterus.

Endometrioid adenocarcinoma is the most frequent form of endometrial cancer, usually developing in pre- and peri-menopausal women. beta-catenin abnormalities are common in endometrioid type endometrial carcinomas with squamous differentiation. To investigate the role of beta-catenin (Ctnnb1) in uterine development and tumorigenesis, mice were generated which expressed a dominant stabilized beta-catenin or had beta-catenin conditionally ablated in the uterus by crossing the PR(Cre) mouse with the Ctnnb1(f(ex3)/+) mouse or Ctnnb1(f/f) mouse, respectively. Both of the beta-catenin mutant mice have fertility defects and the ability of the uterus to undergo a hormonally induced decidual reaction was lost. Expression of the dominant stabilized beta-catenin, PR(cre/+)Ctnnb1(f(ex3)/+), resulted in endometrial glandular hyperplasia, whereas ablation of beta-catenin, PR(cre/+)Ctnnb1(f/f), induced squamous cell metaplasia in the murine uterus. Therefore, we have demonstrated that correct regulation of beta-catenin is important for uterine function as well as in the regulation of endometrial epithelial differentiation.

Enforced epithelial expression of IGF-1 causes hyperplastic prostate growth while negative selection is requisite for spontaneous metastogenesis.

The insulin-like growth factor-1 (IGF-1) signaling axis is important for cell growth, differentiation and survival and increased serum IGF is a risk factor for prostate and other cancers. To study IGF-1 action on the prostate, we created transgenic (PB-Des) mice that specifically express human IGF-1(des) in prostate epithelial cells. This encodes a mature isoform of IGF-1 with decreased affinity for IGF binding proteins (IGFBP) due to a 3-amino acid deletion in the N terminus. Expression of IGF-1(des) was sufficient to cause hyperplastic lesions in all mice, however the well-differentiated lesions did not progress to adenocarcinoma within a year. Remarkably, crossing the PB-Des mice to an established model of prostate cancer delayed progression of organ-confined tumors and emergence of metastatic lesions in young mice. While dissemination of metastatic lesions was widespread in old bigenic mice we did not detect IGF-1(des) in poorly differentiated primary tumors or metastatic lesions. Expression of endogenous IGF-1 and levels of P-Akt and P-Erk were reduced independent of age. These data suggest that increased physiologic levels of IGF-1 facilitate the emergence of hyperplastic lesions while imposing a strong IGF-1-dependent differentiation block. Selection against IGF-1 action appears requisite for progression of localized disease and metastogenesis.

Steroid receptor coactivator 2: an essential coregulator of progestin-induced uterine and mammary morphogenesis.

The importance of the progesterone receptor (PR) in transducing the progestin signal is firmly established in female reproductive and mammary gland biology; however, the coregulators preferentially recruited by PR in these systems have yet to be comprehensively investigated. Using an innovative genetic approach, which ablates gene function specifically in murine cell-lineages that express PR, steroid receptor coactivator 2 (SRC-2, also known as TIF-2 or GRIP-1) was shown to exert potent coregulator properties in progestin-dependent responses in the uterus and mammary gland. Uterine cells positive for PR (but devoid of SRC-2) led to an early block in embryo implantation, a phenotype not shared by knockouts for SRC-1 or SRC-3. In the case of the mammary gland, progestin-dependent branching morphogenesis and alveologenesis failed to occur in the absence of SRC-2, thereby establishing a critical coactivator role for SRC-2 in cellular proliferative programs initiated by progestins in this tissue. Importantly, the recent detection of SRC-2 in both human endometrium and breast suggests that this coregulator may provide a new clinical target for the future management of female reproductive health and/or breast cancer.

Dynamic regulation of progesterone receptor activity in female reproductive tissues.

The progesterone receptor (PR) in cooperation with coregulator complexes coordinates crucial processes in female reproduction. To investigate the dynamic regulation of PR activity in vivo, a new transgenic mouse model utilizing a PR activity indicator (PRAI) system was generated. Studies utilizing the PRAI mouse have revealed that progesterone temporally regulates PR activity in female reproductive tissues. Specifically, progesterone rapidly enhances PR activity immediately after administration. However, chronic progesterone stimulation represses PR activity in female reproductive organs. Like progesterone, RU486 also temporally regulates PR activity in female reproductive organs. However, the temporal regulation of PR activity by RU486 is the inverse of progesterone's activity. RU486 acutely represses PR activity after injection but increases PR activity after chronic treatment in female reproductive tissues. Treatment with a mixed antagonist/agonist of PR, when compared to natural hormone, results in dramatically different tissue-specific patterns of intracellular PR activity, coregulator levels, and kinase activity. Transcriptional regulation of gene expression by PR is facilitated by coordinate interactions with the steroid receptor coactivators (SRCs). Bigenic PRAI-SRC knockout mouse models enabled us to draw a tissue-specific coactivator atlas for PR activity in vivo. Based on this atlas, we conclude that the endogenous physiological function of PR in distinct tissues is modulated by different SRCs. SRC-3 is the primary coactivator for PR in the breast and SRC-1 is the primary coactivator for PR in the uterus.

Expression of an Nkx3.1-CRE gene using ROSA26 reporter mice.

The genetic locus of Nkx3.1, an early murine marker of sclerotome and prostate development, was disrupted by a knock in of CRE recombinase via homologous recombination in embryonic stem cells. Cell fate mapping revealed previously unidentified cell lineages expanded from Nkx3.1-expressing cell populations and recapitulated reported Nkx3.1 expression patterns. In lineage trace experiments of E18.5 Nkx3.1-CRE; R26R embryos novel staining was observed in areas of the lungs, portions of the duodenum, and vertebral elements of the skeleton. beta-galactosidase activity measured in Nkx3.1-CRE; R26R and Nkx3.2-CRE; R26R embryos was observed in overlapping regions of the sclerotome but no apparent change in Nkx3.1 expression was seen in the Nkx3.2 mutants by in situ hybridization.

Molecular mechanisms involved in progesterone receptor regulation of uterine function.

The ovarian steroid hormone progesterone is a major regulator of uterine function. The actions of this hormone is mediated through its cognate receptor, the progesterone receptor, Pgr. Ablation of the Pgr has shown that this receptor is critical for all female reproductive functions including the ability of the uterus to support and maintain the development of the implanting mouse embryo. High density DNA microarray analysis has identified direct and indirect targets of Pgr action. One of the targets of Pgr action is a member of the Hedgehog morphogen Indian Hedgehog, Ihh. Ihh and members of the Hh signaling cascade show a coordinate expression pattern in the mouse uterus during the preimplantation period of pregnancy. The expression of Ihh and its receptor Patched-1, Ptc1, as well as, down stream targets of Ihh-Ptch1 signaling, such as the orphan nuclear receptor COUP-TF II show that this morphogen pathway mediates communication between the uterine epithelial and stromal compartments. The members of the Ihh signaling axis may function to coordinate the proliferation, vascularization and differentiation of the uterine stroma during pregnancy. This analysis demonstrates that progesterone regulates uterine function in the mouse by coordinating the signals from the uterine epithelium to stroma in the preimplantation mouse uterus.

Steroid hormone regulation of Clca3 expression in the murine uterus.

Progesterone (P4) and its cognate receptor, the progesterone receptor (PGR), have important roles in the establishment and maintenance of pregnancy in the murine uterus. In previous studies, using high-density DNA microarray analysis, we identified a subset of genes whose expression is repressed by chronic P4-PGR activation in the uterus. The Clca3 gene is one of the genes whose expression is the most significantly downregulated by P4 and PGR. In the present study, we performed real-time RT-PCR and in situ hybridization to investigate the regulation of Clca3 by P4 and determine the pattern of expression of Clca3 in the uterus during early pregnancy. This analysis shows that Clca3 mRNA transcripts were detected in the luminal and glandular epithelium of the pseudopregnant uterus at day 0.5 and that the expression of Clca3 was not detected after day 3.5. P4 represses Clca3 mRNA synthesis in the luminal epithelial and glandular epithelial cells of the uterus in ovariectomized wild-type mice, but not in Pgr knockout (PRKO) mice. Conversely, estrogen (E2) induces Clca3 expression in the luminal epithelium and glandular epithelium, and this induction was repressed by P4 in the murine uterus. Analysis of the promoter region of Clca3 by in silico and transient transfection analysis in HEC-1A cells identified the regulation of Clca3 by estrogen receptor-alpha (ESR1) within the first 528 bp of 5'-flanking region of the Clca3 gene. Our studies identified Clca3 as a novel downregulated gene of PGR that is a direct target of E2 regulation.

Functional and clinical characterization of a mutation in KCNJ2 associated with Andersen-Tawil syndrome.

BACKGROUND: Andersen-Tawil syndrome (ATS) is a rare inherited disorder, characterised by periodic paralysis, cardiac dysarrhythmias, and dysmorphic features, and is caused by mutations in the gene KCNJ2, which encodes the inward rectifier potassium channel, Kir2.1. This study sought to analyse KCNJ2 in patients with familial ATS and to determine the functional characteristics of the mutated gene. METHODS AND RESULTS: We screened a family with inherited ATS for the mutation in KCNJ2, using direct DNA sequencing. A missense mutation (T75R) of Kir2.1, located in the highly conserved cytoplasmic N-terminal domain, was identified in three affected members of this family. Using the Xenopus oocyte expression system and whole cell voltage clamp analyses, we found that the T75R mutant was non-functional and possessed a strong dominant negative effect when co-expressed with the same amount of wild type Kir2.1. Transgenic (Tg) mice expressing the mutated form of Kir2.1 in the heart had prolonged QTc intervals compared with mice expressing the wild type protein. Ventricular tachyarrhythmias were observed in 5 of 14 T75R-Tg mice compared with 1 of 7 Wt-Tg and none of 6 non-transgenic littermates. In three of five T75R-Tg mice with ventricular tachycardia, their ECG disclosed bidirectional tachycardia as in our proband. CONCLUSIONS: The in vitro studies revealed that the T75R mutant of Kir2.1 had a strong dominant negative effect in the Xenopus oocyte expression system. It still preserved the ability to co-assemble and traffic to the cell membrane in mammalian cells. For in vivo studies, the T75R-Tg mice had bidirectional ventricular tachycardia after induction and longer QT intervals.

Alterations in glucose homeostasis in SSTR1 gene-ablated mice.

SSTR1 is found on the majority of human pancreatic beta cells, however, its role in insulin secretion has yet to be elucidated. In this study, we used the SSTR1 knockout mouse model to examine the role of SSTR1 in insulin secretion and glucose homeostasis in mice. Despite the reported effect of SSTR1 in inhibiting growth hormone secretion, SSTR1-/- mice had significantly reduced body weight with growth retardation. Perfusion of isolated mouse pancreata at 3 months of age demonstrated a significant increase in insulin secretion in SSTR1-/- mice compared with that of WT controls. We also found that at 3 months of age, SSTR1-/- mice had significantly decreased levels of systemic insulin secretion and were glucose intolerant. However, SSTR1 gene-ablated mice had a much higher rate of insulin clearance compared to WT mice at the same age. When challenged at 12 months of age, we found SSTR1-/- mice had increased glucose tolerance with exaggerated increase of insulin levels at the end of the experiment. Immunochemical analysis showed that the pancreatic islets of SSTR1-/- mice had significantly decreased levels of somatostatin staining and a significant decrease of SSTR5 expression. These results demonstrate that SSTR1 plays an important role in the regulation of insulin secretion in the endocrine pancreas in mice.

The effect of global SSTR5 gene ablation on the endocrine pancreas and glucose regulation in aging mice.

INTRODUCTION: The purpose of this study was to examine the effect of global gene ablation of SSTR5 on the endocrine pancreas, insulin secretion, and glucose tolerance in aging mice, as SSTR5 is a primary regulator of insulin secretion in the mouse pancreas. METHODS: Global SSTR5-/- mice were generated and genotypes were verified using Southern blot and RT-PCR. Glucose tolerance and in vivo insulin secretion in SSTR5-/- and WT mice were examined using intraperitoneal glucose tolerance test (IPGTT;1.2-2.0 mg/kg) at 3 and 12 months of age (n = 8 per group). Basal and glucose-stimulated insulin secretion in vitro was studied using the isolated perfused mouse pancreas model at 3 and 12 months. Pancreata were removed and levels of insulin, glucagon, somatostatin, and SSTR1 were studied using immunohistochemical analysis along with H&E staining of the pancreata. RESULTS: Genotyping verified the absence of SSTR5 in SSTR5-/- mice. IPGTT demonstrated that 3-month-old SSTR5-/- mice were glucose intolerant despite similar insulin secretion both in vivo and in vitro and enlarged islets. At 12 months of age, SSTR5-/- mice had basal hypoglycemia and improved glucose intolerance associated with hyperinsulinemia in vivo and in vitro and enlarged islets. SSTR5-/- mice had increased insulin clearance at 3 and 12 months of age. SSTR1 expression was significantly increased in islets at 3 months of age, but was nearly absent in islets at 12 months of age, as was somatostatin staining in SSTR5-/- mice. CONCLUSIONS: These results suggest that both SSTR5 and SSTR1 play a pivotal role in insulin secretion and glucose regulation in mice and that their regulatory effects are age-related.

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice.

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.

Double-gene ablation of SSTR1 and SSTR5 results in hyperinsulinemia and improved glucose tolerance in mice.

BACKGROUND: Previous studies conducted in our laboratory showed that single-gene ablation of somatostatin receptor (SSTR)1 or 5 results in diabetes in mice. The objective of this study was to determine the effect of double-gene ablation of SSTR1 and SSTR5 on insulin secretion and glucose homeostasis in mice. METHODS: SSTR1/5 -/- mice and wild-type (WT) control mice were generated and their genotype verified via polymerase chain reaction. Insulin secretion and glucose levels in these mice were examined with the use of an intraperitoneal glucose tolerance test (1.2-2.0 g/kg body weight). In vitro glucose-stimulated insulin secretion was studied with the use of the isolated perfused mouse pancreas model and islet culture techniques. Pancreata morphologic alterations were determined, and an immunohistochemistry analysis was performed. RESULTS: In vitro incubation of isolated islets from WT mice with somatostatin peptides resulted in significant reduction in insulin secretion, whereas SSTR1/5 -/- mouse islets had no response to somatostatin peptides confirming SSTR1/5 gene ablation. SSTR1/5 -/- mice also had significant increase of both basal and glucose-stimulated insulin levels in vitro. During the intraperitoneal glucose tolerance test, SSTR1/5 -/- mice had significantly improved glucose tolerance and sustained an increase in late-phase insulin secretion in vivo. Histological analysis demonstrated significant islet hyperplasia in the SSTR 1/5 -/- mouse pancreas. Immunostaining revealed an overall increase of glucagon and pancreatic polypeptide-producing cells in the islets of SSTR1/5 -/- mice. CONCLUSIONS: Double-gene ablation of SSTR1 and SSTR5 in mice resulted in a distinct phenotype with islet cell hyperplasia, hyperinsulinemia, and improved glucose tolerance. This form of diabetes differs from that seen in mice in which only the SSTR1 or SSTR5 gene was ablated. These results demonstrate that SSTR1 and SSTR5 are important regulators of insulin secretion and glucose regulation, and suggest that SSTR1 and SSTR5 are coordinately regulated.

Progesterone induction of calcitonin expression in the murine mammary gland.

Progesterone, via its nuclear receptor, is mandatory not only for the induction and specification of mammary gland ductal side-branching and lobuloalveologenesis but also for carcinogen-induced mammary tumorigenesis. Notwithstanding these recent advances, a more comprehensive molecular explanation of progesterone-induced mammary morphogenesis is contingent upon the identification and characterization of mammary molecular targets that are responsive to the progesterone signal. Toward this goal, we report that calcitonin, a 32 amino acid peptide hormone involved in calcium homeostasis, is exclusively expressed in, and secreted from, luminal epithelial cells within the mammary gland of the pregnant mouse, and, importantly, its expression is progesterone-dependent. Conversely, the calcitonin receptor is present during all stages of post-natal mammary development examined, is localized to the myoepithelial cell lineage, and is not regulated by progesterone. Because calcitonin induction spatiotemporally correlates with increases in progesterone-induced mammary gland proliferation and structural remodeling, we posit that calcitonin - through its receptor - may be involved in one or both of these progesterone-dependent processes.

Genetically engineered mouse models for lung cancer.

The lung is a complex organ consisting of numerous cell types that function to ensure sufficient gas exchange to oxygenate the blood. In order to accomplish this function, the lung must be exposed to the external environment and at the same time maintain a homeostatic balance between its function in gas exchange and the maintenance of inflammatory balance. During the past two decades, as molecular methodologies have evolved with the sequencing of entire genomes, the use of in vivo models to elucidate the molecular mechanisms involved in pulmonary physiology and disease have increased. The mouse has emerged as a potent model to investigate pulmonary physiology due to the explosion in molecular methods that now allow for the developmental and tissue-specific regulation of gene transcription. Initial efforts to manipulate gene expression in the mouse genome resulted in the generation of transgenic mice characterized by the constitutive expression of a specific gene and knockout mice characterized by the ablation of a specific gene. The utility of these original mouse models was limited, in many cases, by phenotypes resulting in embryonic or neonatal lethality that prevented analysis of the impact of the genetic manipulation on pulmonary biology. Second-generation transgenic mouse models employ multiple strategies that can either activate or silence gene expression thereby providing extensive temporal and spatial control of the experimental parameters of gene expression. These highly regulated mouse models are intended to serve as a foundation for further investigation of the molecular basis of human disease such as tumorigenesis. This review describes the principles, progress, and application of systems that are currently employed in the conditional regulation of gene expression in the investigation of lung cancer.

Multiple mechanisms for oxygen-induced regulation of the Clara cell secretory protein gene.

The Clara cell secretory protein (CCSP) imparts a protective effect to the lung during oxidant injury. However, exposure to supplemental oxygen, a common therapeutic modality for lung disease, represses the expression of CCSP in the adult mouse lung. We investigated the mechanisms of hyperoxia-induced repression of the mouse CCSP promoter. Deletion experiments in vivo and in vitro indicated that the hyperoxia-responsive elements are localized to the proximal -166 bp of the CCSP promoter. Electrophoretic mobility shift and supershift analyses demonstrated increased binding of c-Jun at the activator protein-1 site, increased binding of CCAAT/enhancer binding protein (C/EBP) beta at the C/EBP sites, and decreased binding at the Nkx2.1 sites. Western analyses revealed that hyperoxia exposure induced an increase in the expression of the C/EBPbeta isoform liver-inhibiting protein (LIP) and an increase in cytoplasmic Nkx2.1. Cotransfection of LIP or c-Jun expression plasmids decreased the transcriptional activity of the proximal -166-bp CCSP promoter. These observations suggest that hyperoxia-induced repression of the CCSP gene is mediated, at least in part, at the level of transcription and that multiple mechanisms mediate this repression. Moreover, these novel observations may provide insights for generation of therapeutic interventions for the amelioration of oxidant-induced lung injury.