Transient Transgenics: An Efficient Method to Identify Gene Regulatory Elements.

We describe a novel, efficient method to identify cis-acting DNA sequences that drive cell-specific gene expression during development. We utilize transfer of Bacterial Artificial Chromosome (BAC) genomic DNAs, modified to contain a reporter gene, into fertilized mouse embryos and placing the injected embryos into pseudopregnant recipient females. The embryos are allowed to develop in utero for defined times after which they are collected for analysis. Using DNAs containing the LacZ reporter gene facilitates the analysis of gene activity through microscopy of intact embryos and subsequent sectioning of the stained embryos. With this technique cis-element activity can be identified and evaluated through further mutational analysis of the injected BAC DNA. This allows the identification of important gene regulatory domains that specify stage-specific gene expression in the developing embryo.

Mesenchyme-derived IGF2 is a major paracrine regulator of pancreatic growth and function.

The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Imprinted genes, which are expressed in a parent-of-origin-specific manner, are known to have important roles in development, growth and metabolism. However, our knowledge regarding their roles in the control of pancreatic growth and function remains limited. Here we show that many imprinted genes are highly expressed in pancreatic mesenchyme-derived cells and explore the role of the paternally-expressed insulin-like growth factor 2 (Igf2) gene in mesenchymal and epithelial pancreatic lineages using a newly developed conditional Igf2 mouse model. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of any discernible growth or functional phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Additionally, increased IGF2 levels specifically in the mesenchyme, through conditional Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Furthermore, ex-vivo exposure of primary acinar cells to exogenous IGF2 activates AKT, a key signalling node, and increases their number and amylase production. Based on these findings, we propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.

Synergistic immunologic targets for the treatment of prostate cancer.

Prostate cancer is a common disease and, while detection and treatment have advanced, it remains a significant cause of morbidity and mortality in men. Research suggests significant involvement of the immune system in the pathogenesis and progression of prostate cancer, indicating that immunologic therapies may benefit patients. Two immunologic factors, interleukin-2 and transforming growth factor-ß, may be especially attractive therapeutic targets for prostate cancer. Specifically, an increase in interleukin-2 signaling and a decrease in transforming growth factor-ß signaling might help improve immunologic recognition and targeting of tumor cells. The purpose of this review is to highlight the evidence that interleukin-2 and blockade of transforming growth factor-ß could be used to target prostate cancer based on current understanding of immune function in the context of prostate cancer. Additionally, current treatments related to these two factors for prostate and other cancers will be used to strengthen the argument for this strategy.

Spectrin's chimeric E2/E3 enzymatic activity.

In this minireview, we cover the discovery of the human erythrocyte α spectrin E2/E3 ubiquitin conjugating/ligating enzymatic activity and the specific cysteines involved. We then discuss the consequences when this activity is partially inhibited in sickle cell disease and the possibility that the same attenuation is occurring in multiple organ dysfunction syndrome. We finish by discussing the reasons for believing that nonerythroid α spectrin isoforms (I and II) also have this activity and the importance of testing this hypothesis. If correct, this would suggest that the nonerythroid spectrin isoforms play a major role in protein ubiquitination in all cell types. This would open new fields in experimental biology focused on uncovering the impact that this enzymatic activity has upon protein-protein interactions, protein turnover, cellular signaling, and many other functions impacted by spectrin, including DNA repair.

New concepts concerning prostate cancer screening.

Prostate Cancer (CaP) is rapidly becoming a worldwide health issue. While CaP mortality has decreased in recent years, coincident with the widespread use of Prostate-Specific Antigen (PSA) screening, it remains the most common solid tumor in men and is the second leading cause of cancer death in the United States. The frequency of CaP is growing not only in western cultures, but also its incidence is dramatically increasing in eastern nations. Recently, examination of data from long-term trials and follow up has cast a shadow on the effectiveness of employing PSA as a primary screening tool for CaP. In this review, we not only summarize opinions from this examination and synthesize recommendations from several groups that suggest strategies for utilizing PSA as a tool, but also call for research into biomarkers for CaP diagnosis and disease progression. We also describe our recent work that identified a smooth muscle contractile protein in prostate epithelia, namely smooth muscle gamma actin, and indicate the potential for this molecule as a new unique footprint and as a CaP marker.

Pancreatic mesenchyme regulates epithelial organogenesis throughout development.

The developing pancreatic epithelium gives rise to all endocrine and exocrine cells of the mature organ. During organogenesis, the epithelial cells receive essential signals from the overlying mesenchyme. Previous studies, focusing on ex vivo tissue explants or complete knockout mice, have identified an important role for the mesenchyme in regulating the expansion of progenitor cells in the early pancreas epithelium. However, due to the lack of genetic tools directing expression specifically to the mesenchyme, the potential roles of this supporting tissue in vivo, especially in guiding later stages of pancreas organogenesis, have not been elucidated. We employed transgenic tools and fetal surgical techniques to ablate mesenchyme via Cre-mediated mesenchymal expression of Diphtheria Toxin (DT) at the onset of pancreas formation, and at later developmental stages via in utero injection of DT into transgenic mice expressing the Diphtheria Toxin receptor (DTR) in this tissue. Our results demonstrate that mesenchymal cells regulate pancreatic growth and branching at both early and late developmental stages by supporting proliferation of precursors and differentiated cells, respectively. Interestingly, while cell differentiation was not affected, the expansion of both the endocrine and exocrine compartments was equally impaired. To further elucidate signals required for mesenchymal cell function, we eliminated ß-catenin signaling and determined that it is a critical pathway in regulating mesenchyme survival and growth. Our study presents the first in vivo evidence that the embryonic mesenchyme provides critical signals to the epithelium throughout pancreas organogenesis. The findings are novel and relevant as they indicate a critical role for the mesenchyme during late expansion of endocrine and exocrine compartments. In addition, our results provide a molecular mechanism for mesenchymal expansion and survival by identifying ß-catenin signaling as an essential mediator of this process. These results have implications for developing strategies to expand pancreas progenitors and ß-cells for clinical transplantation.

Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle alpha-actin gene.

Smooth muscle alpha actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full-length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1-3 copies of the mCherry-substituted BAC vector. Furthermore, we characterized the expression of SMA-mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence-activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA-expressing cells via FACS and for studying the emergence, behavior, and regulation of SMA-expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development.

Intronic DNA elements regulate androgen-dependent expression of the murine Nkx3.1 gene.

Nkx3.1 is a well-conserved homeobox gene that is involved in development, differentiation and maintenance of prostate epithelial cells. Nkx3.1 expression is induced by androgen in prostate epithelia and, as such, our interest is to understand the mechanism(s) for this androgen-dependent expression in normal epithelial cells. In this report, we show that the region of DNA sequence 2.7 kilobases in front of the mouse Nkx3.1 gene drives enhanced transcription in prostate epithelia cells; however, this segment was not capable of androgen-directed regulation. Among the multiple, potential androgen response elements (AREs) identified by scanning sequences near and within the gene, two sequences within the intron of the murine Nkx3.1 gene were demonstrated to confer androgen-dependent transcription in reporter gene transfection experiments. Each of the elements, termed ARE A and ARE B, contained a 6-base pair core sequence, TGTTCT, that has been described as an androgen receptor half-site binding sequence, separated by 498 base pairs of DNA. Both of the intronic half-sites bind activated androgen receptor from a variety of sources, albeit with different apparent affinities. This region of the Nkx3.1 gene demonstrates a high degree of conservation among diverse species and mutagenesis experiments demonstrated that both elements are required for androgen stimulation. Taken together, our study shows that androgen-dependent transcription of the mouse Nkx3.1 gene is conferred through a noncanonical element within the intron of the gene.

Myocardin-dependent activation of the CArG box-rich smooth muscle gamma-actin gene: preferential utilization of a single CArG element through functional association with the NKX3.1 homeodomain protein.

Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds a 10-bp element known as the CArG box, located in the proximal regulatory region of hundreds of target genes. SRF activates target genes in a cell- and context-dependent manner by assembling unique combinations of cofactors over CArG elements. One particularly strong SRF cofactor, myocardin (MYOCD), acts as a component of a molecular switch for smooth muscle cell (SMC) differentiation by activating cytoskeletal and contractile genes harboring SRF-binding CArG elements. Here we report that the human ACTG2 promoter, containing four conserved CArG elements, displays SMC-specific basal activity and is highly induced in the presence of MYOCD. Stable transfection of a non-SMC cell type with Myocd elicits elevations in endogenous Actg2 mRNA. Gel shift and luciferase assays reveal a strong bias for MYOCD-dependent transactivation through CArG2 of the human ACTG2 promoter. Substitution of CArG2 with other CArGs, including a consensus CArG element, fails to reconstitute full MYOCD-dependent ACTG2 promoter stimulation. Mutation of an adjacent binding site for NKX3.1 reduces MYOCD-dependent transactivation of the ACTG2 promoter. Co-immunoprecipitation, glutathione S-transferase pulldown, and luciferase assays show a physical and functional association between MYOCD and NKX3.1; no such functional relationship is evident with the related NKX2.5 transcription factor despite its interaction with MYOCD. These results demonstrate the ability of MYOCD to discriminate among several juxtaposed CArG elements, presumably through its novel partnership with NKX3.1, to optimally transactivate the human ACTG2 promoter.

Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development.

BACKGROUND & AIMS: Expansion and patterning of the endoderm generate a highly ordered, multiorgan digestive system in vertebrate animals. Among distal foregut derivatives, the gastric corpus, antrum, pylorus, and duodenum are distinct structures with sharp boundaries. Some homeodomain transcription factors expressed in gut mesenchyme convey positional information required for anterior-posterior patterning of the digestive tract. Barx1, in particular, controls stomach differentiation and morphogenesis. The Nirenberg and Kim homeobox gene Bapx1 (Nkx3-2) has an established role in skeletal development, but its function in the mammalian gut is less clear. METHODS: We generated a Bapx1(Cre) knock-in allele to fate map Bapx1-expressing cells and evaluate its function in gastrointestinal development. RESULTS: Bapx1-expressing cells populate the gut mesenchyme with a rostral boundary in the hindstomach near the junction of the gastric corpus and antrum. Smooth muscle differentiation and distribution of early regional markers are ostensibly normal in Bapx1(Cre/Cre) gut, but there are distinctive morphologic abnormalities near this rostral Bapx1 domain: the antral segment of the stomach is markedly shortened, and the pyloric constriction is lost. Comparison of expression domains and examination of stomach phenotypes in single and compound Barx1 and Bapx1 mutant mice suggests a hierarchy between these 2 factors; Bapx1 expression is lost in the absence of Barx1. CONCLUSIONS: This study reveals the nonredundant requirement for Bapx1 in distal stomach development, places it within a Barx1-dependent pathway, and illustrates the pervasive influence of gut mesenchyme homeobox genes on endoderm differentiation and digestive organogenesis.

Smooth muscle-specific gene delivery in the vasculature based on restriction of DNA nuclear import.

The two currently employed approaches restricting gene delivery and/or expression to desired cell types in vivo rely on cell surface targeting or cell-specific promoters. We have developed a third approach based on cell-specific nuclear transport of the delivered plasmid DNA. We have previously shown that plasmid nuclear import in non-dividing cells is sequence-specific and have identified a set of cell-specific DNA nuclear targeting sequences that can be used to limit DNA nuclear import to desired cell types. Specifically we have identified elements of the smooth muscle gamma actin (SMGA) promoter that direct plasmid nuclear import selectively in smooth muscle cells (SMCs) in vitro (Vacik et al, 1999, Gene Therapy 6:1006-1014). In the present study, we demonstrate that the SMC-specific DNA nuclear targeting sequence from the SMGA promoter drives nuclear accumulation of plasmids and subsequent gene expression exclusively in the smooth muscle cell layer of the vessel wall in the intact vasculature of rats using electroporation mediated delivery. These results demonstrate that certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types providing a new, novel means of cell targeting for gene therapy.

Structural and functional analysis of domains mediating interaction between the bagpipe homologue, Nkx3.1 and serum response factor.

Nkx3.1 is a member of the NK2 class of homeodomain proteins and is expressed in development, being an early marker of the sclerotome and prostate gland. It has been shown to be a critical factor for prostate differentiation and function. Previous studies suggested that Nkx3.1 interacts with Serum Response Factor (SRF) to transactivate the Smooth Muscle gamma-Actin (SMGA) promoter. In studies presented here, we examined the molecular mechanisms underlying the functional synergy of these factors upon SMGA transcription. We demonstrate that full length Nkx3.1 physically interacts with SRF in the absence of DNA and that these factors are able to co-associate in cellular context using a mammalian two-hybrid system. The segment of SRF responsible for Nkx3.1 interaction was mapped to a approximately 30 amino acid region (AAs 142-171) at the N-terminal segment of the MADS box. Two separate regions of Nkx3.1 were found to mediate interactions with SRF. Interestingly, recognized domains of NK2 proteins, namely the TN, homeodomain DNA binding segment, and the NK2-SD do not participate in SRF interactions. One of the Nkx3.1 SRF binding domains was mapped to the N-terminal of the protein consistent with recent studies of these proteins using NMR spectroscopy by Gelmann and colleagues (1). A second SRF binding region was mapped to amino acids C-terminal to the homeodomain. Structural predictions indicate that both of the SRF interacting segments are largely hydrophobic in character and beta-strand in structure. With co-transfection transcriptional analyses we found that interaction between SRF and Nkx3.1 as well as DNA binding by both factors was required for the observed transcriptional synergy. Thus our studies have identified novel protein-protein interacting domains within Nkx3.1 and SRF that operate in concert with their respective DNA binding domains to mediate functional transcriptional synergy of these factors to regulate SMGA gene activation.

Promoter methylation is associated with the age-dependent loss of N-cadherin in the rat kidney.

The cadherins are cell adhesion molecules required for cellular homeostasis, and N-cadherin is the predominant cadherin expressed in proximal tubular epithelial cells in humans and rats. Our laboratory previously reported an age-dependent decrease in renal N-cadherin expression; the levels of N-cadherin mRNA and protein expression decreased in parallel, implicating a transcriptional mechanism in the age-dependent loss of expression (19). In this study, we examined the hypothesis that promoter hypermethylation underlies the loss of N-cadherin expression in aging rat kidney. We cloned the 5' flanking region of the rat N-cadherin gene and observed basic promoter activity in a 3,992-bp region localized immediately upstream of the ATG start site. Nucleotide analysis revealed 87% identity with the human N-cadherin minimal promoter region. Consistent with a role for regulation by DNA methylation, we found that a dense CpG island, which spans 1,104 bp (-1,158 to -55), flanks the rat N-cadherin gene; a similar CpG profile was found in the human N-cadherin 5' flanking region. Methylation-specific PCR analysis demonstrated that the promoter region of N-cadherin is heavily methylated in aged, but not young, rat kidney. Interestingly, the promoter is not methylated in age-matched, calorically restricted animals. In contrast, the promoter region is not methylated in either young or aged rat liver; this corresponds to the finding that aging is not associated with decreased N-cadherin expression in the liver. In addition, N-cadherin expression is markedly induced in NRK-52E cells treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, further suggesting that methylation at CpG in the promoter region may underlie the age-dependent decrease in renal N-cadherin expression.

Immunohistochemical localization of cadherin and catenin adhesion molecules in the murine growth plate.

Mouse tibial growth plates were examined for the presence of adhesion molecules using immunohistochemistry and RT-PCR. All of the components of the classical cadherin/catenin complex (cadherin, alpha-, beta-, and gamma-catenin), as well as a heavy presence of p120, were identified in the murine growth plate. All of the major cadherins (1-5, 11, 13, and 15) were, for the first time, identified and localized in the murine growth plate. We have demonstrated that most of the cadherins and catenins reside in the zone of hypertrophy. Only alpha-catenin and E-, P-, R-, and VE-cadherin were found in all regions of the growth plate. The results for T-cadherin were inconclusive.

COUP-TFII is essential for radial and anteroposterior patterning of the stomach.

COUP-TFII, an orphan member of the steroid receptor superfamily, has been implicated in mesenchymal-epithelial interaction during organogenesis. The generation of a lacZ knock-in allele in the COUP-TFII locus in mice allows us to use X-gal staining to follow the expression of COUP-TFII in the developing stomach. We found COUP-TFII is expressed in the mesenchyme and the epithelium of the developing stomach. Conditional ablation of floxed COUP-TFII by Nkx3-2Cre recombinase in the gastric mesenchyme results in dysmorphogenesis of the developing stomach manifested by major patterning defects in posteriorization and radial patterning. The epithelial outgrowth, the expansion of the circular smooth muscle layer and enteric neurons as well as the posteriorization of the stomach resemble phenotypes exhibited by inhibition of hedgehog signaling pathways. Using organ cultures and cyclopamine treatment, we showed downregulation of COUP-TFII level in the stomach, suggesting COUP-TFII as a target of hedgehog signaling in the stomach. Our results are consistent with a functional link between hedgehog proteins and COUP-TFII, factors that are vital for epithelial-mesenchymal interactions.

Recruitment of the androgen receptor via serum response factor facilitates expression of a myogenic gene.

Androgen receptor (AR) induced precocious myogenesis in culture and myogenic specified gene activity. Increased levels of AR expression in replicating C2C12 myoblasts stimulated fusion into post-differentiated multinucleated myotubes and the appearance of skeletal alpha-actin transcripts, even in the absence of ligand. Furthermore, AR activated the skeletal alpha-actin promoter, which lacks GRE sites, in co-transfected C2C12 cells. AR co-activation of the skeletal alpha-actin promoter required co-expressed full-length serum response factor (SRF). In vitro, AR associated with SRF and was recruited by SRF to a alpha-actin promoter SRF binding site. Our data suggest that AR is capable of activating myogenic genes devoid of consensus AR binding sites via its recruitment by the myogenic enriched transcription factor, SRF.

Transforming growth factor-beta induction of smooth muscle cell phenotpye requires transcriptional and post-transcriptional control of serum response factor.

Transforming growth factor-beta induces a smooth muscle cell phenotype in undifferentiated mesenchymal cells. To elucidate the mechanism(s) of this phenotypic induction, we focused on the molecular regulation of smooth muscle-gamma-actin, whose expression is induced at late stages of smooth muscle differentiation and developmentally restricted to this lineage. Transforming growth factor-beta induced smooth muscle-gamma-actin protein, cytoskeletal localization, and mRNA expression in mesenchymal cells. Smooth muscle-gamma-actin promoter-luciferase reporter activity was enhanced by transforming growth factor-beta, and deletion analysis revealed that CArG box 2 in the promoter was necessary for this transcriptional activation. CArG motifs bind transcriptional activator serum response factor; gel shift analyses revealed increased binding of serum response factor-containing complexes to this site in response to transforming growth factor-beta, paralleled by increased serum response factor protein expression. Serum response factor expression was found to be up-regulated by transforming growth factor-beta via transcriptional activation of the gene and post-transcriptional regulation. Using mesenchymal cells stably transfected with wild type or dominant-negative serum response factor, we demonstrated that its expression is sufficient for induction of a smooth muscle phenotype in mesenchymal cells and is necessary for transforming growth factor-beta-mediated smooth muscle induction.