A new regulatory region of the IL-2 locus that confers position-independent transgene expression

Although the promoter/enhancer of the IL-2 gene mediates inducible reporter gene expression in vitro, it cannot drive consistent expression in transgenic mice. The location and existence of any regulatory elements that could open the IL-2 locus in vivo have remained unknown, preventing analysis of IL-2 regulation in developmental contexts. In this study, we report the identification of such a regulatory region, marked by novel DNase-hypersensitive sites upstream of the murine IL-2 promoter in unstimulated and stimulated T cells. Inclusion of most of these sites in an 8.4-kb IL-2 promoter green fluorescent protein transgene gives locus control region-like activity. Expression is efficient, tissue specific, and position independent. This transgene is expressed not only in peripheral T cells, but also in immature thymocytes and thymocytes undergoing positive selection, in agreement with endogenous IL-2 expression. In contrast, a 2-kb promoter green fluorescent protein transgene, lacking the new hypersensitive sites, is expressed in only a few founder lines, and expression is dysregulated in CD8(+) cells. Thus, the 6.4 kb of additional upstream IL-2 sequence contains regulatory elements that provide integration site independence and differential regulation of transgene expression in CD8 vs CD4 cells.     

A reporter transgene based on a human keratin 6 gene promoter is specifically expressed in the periderm of mouse embryos

We report the developmental regulation of a lacZ reporter transgene fused to the promoter region of the human keratin 6a gene. In mouse embryos, the transgene is expressed in the periderm (the outermost layer of embryonic epidermis), as are the endogenous keratin 6 alpha and beta genes. A subset of periderm cells, localized to temporary epithelial fusions, is known to contain keratin 6 protein, and we find that these cells also harbor LacZ enzymatic activity.     

Expression and function of connexins in the epidermis, analyzed with transgenic mouse mutants

Eight different connexins are expressed in mouse epidermis with overlapping expression patterns in different epidermal layers. Analyses of mice with deficiency or modifications of distinct connexins yielded insights into the large variety of connexins in the epidermis. Connexin43 (Cx43) deficiency in mouse epidermis resulted in a significant acceleration of wound closure. Truncation by 125 amino acid residues of the Cx43 C-terminal region led to an altered epidermal expression pattern of Cx43 and defective development of the epidermal water barrier in transgenic mice, although the truncated Cx43 protein could still form open gap junctional channels in transfected HeLa cells. Thus, the phenotypic abnormalities observed in mice with truncated Cx43 protein (Cx43K258Stop) are more likely due to defective regulation of this protein rather than the closed Cx43 channel. Our studies of connexin-deficient mice revealed an extensive redundancy of connexins expressed in mouse epidermis. Epidermal connexins seem to form two functional groups in which deficiency of one connexin isoform can be compensated by other connexin isoforms of the same group.     

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.     

Generation and evaluation of an IPTG-regulated version of Vav-gene promoter for mouse transgenesis

Different bacteria-derived systems for regulatable gene expression have been developed for the use in mammalian cells and some were also successfully adopted for in vivo use in vertebrate model organisms. However, certain limitations apply to most of these systems, including leakiness of transgene expression, inefficient transgene silencing or activation, as well as limited tissue accessibility of transgene-inducers or their unfavourable pharmacokinetics. In this study, we evaluated the suitability of the lac-operon/lac-repressor (lacO/lacI) system for the regulation of the well-established Vav-gene promoter that allows inducible transgene expression in different haematopoietic lineages in mice. Using the fluorescence marker protein Venus as a reporter, we observed that the lacO/lacI system could be amended to modulate transgene-expression in haematopoietic cells. However, reporter expression was not uniform and the lacO elements introduced into the Vav-gene promoter only conferred limited repression and reversion of lacI-mediated gene silencing after administration of IPTG. Although further optimization of the system is required, the lacO-modified version of the Vav-gene promoter may be adopted as a tool where low basal gene-expression and limited transient induction of protein expression are desired, e.g. for the activation of oncogenes or transgenes that act in a dominant-negative manner.     

Characterization of loricrin regulation in vitro and in transgenic mice

We have previously shown that the promoter of a 6.5 kb mouse loricrin clone contains a functional AP-1 element and directs tissue-specific, but not differentiation-specific, expression. We now report the isolation of a 14-kb genomic clone containing an additional 7 kb of genomic sequence. The additional sequences limit expression of a reporter construct to differentiated keratinocytes in culture. The expression of the 6.5-kb and 14-kb loricrin constructs were also analyzed in transgenic mice. Significantly, loricrin was found in all layers of the epidermis of the 6.5-kb transgenics, including basal and spinous cells. The expression of the 14-kb clone was indistinguishable from that of the endogenous gene, confirming that the additional sequences contain negative regulatory elements that restrict loricrin expression to the granular layer in vivo. In addition, we show the AP-1 element localized in the loricrin proximal promoter is necessary but not sufficient for expression of the loricrin gene in vivo in transgenic mice. Finally, to gain further insight into how AP-1 family members regulate expression of the loricrin gene, we co-transfected the loricrin reporter constructs with expression plasmids for various fos and jun family members and demonstrated that c-Fos/Jun-B heterodimers could mimic the differentiation-specific induction of loricrin.     

Evidence of finely tuned expression of DNA polymerase beta in vivo using transgenic mice

DNA polymerase (Pol) is an error-prone repair DNA polymerase that has been shown to create genetic instability and tumorigenesis when overexpressed by only 2-fold in cells, suggesting that a rigorous regulation of its expression may be essential in vivo. To address this question, we have generated mice which express a transgene (Tg) bearing the Pol cDNA under the control of the ubiquitous promoter of the mouse H-2K gene from the major histocompatibility complex. These mice express the Tg only in thymus, an organ which normally contains the most abundant endogenous Pol mRNA and protein, supporting the idea of a tight regulation of Pol in vivo. Furthermore, we found no tumor incidence, suggesting that the single Pol overexpression event is not sufficient to initiate tumorigenesis in vivo.     

Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway

The epidermis comprises multiple layers of specialized epithelial cells called keratinocytes. As cells are lost from the outermost epidermal layers, they are replaced through terminal differentiation, in which keratinocytes of the basal layer cease proliferating, migrate upwards, and eventually reach the outermost cornified layers. Normal homeostasis of the epidermis requires that the balance between proliferation and differentiation be tightly regulated. The GTP binding protein RhoA plays a fundamental role in the regulation of the actin cytoskeleton and in the adhesion events that are critically important to normal tissue homeostasis. Two central mediators of the signals from RhoA are the ROCK serine/threonine kinases ROCK-I and ROCK-II. We have analyzed ROCK's role in the regulation of epidermal keratinocyte function by using a pharmacological inhibitor and expressing conditionally active or inactive forms of ROCK-II in primary human keratinocytes. We report that blocking ROCK function results in inhibition of keratinocyte terminal differentiation and an increase in cell proliferation. In contrast, activation of ROCK-II in keratinocytes results in cell cycle arrest and an increase in the expression of a number of genes associated with terminal differentiation. Thus, these results indicate that ROCK plays a critical role in regulating the balance between proliferation and differentiation in human keratinocytes.     

Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector

To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1beta, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.     

sPLA2 and the epidermal barrier

The mammalian epidermis provides both an interface and a protective barrier between the organism and its environment. Lipid, processed into water-impermeable bilayers between the outermost layers of the epidermal cells, forms the major barrier that prevents water from exiting the organism, and also prevents toxins and infectious agents from entering. The secretory phospholipase 2 (sPLA2) enzymes control important processes in skin and other organs, including inflammation and differentiation. sPLA2 activity contributes to epidermal barrier formation and homeostasis by generating free fatty acids, which are required both for formation of lamellar membranes and also for acidification of the stratum corneum (SC). sPLA2 is especially important in controlling SC acidification and establishment of an optimum epidermal barrier during the first postnatal week. Several sPLA2 isoforms are present in the epidermis. We find that two of these isoforms, sPLA2 IIA and sPLA2 IIF, localize to the upper stratum granulosum and increase in response to experimental barrier perturbation. sPLA2F^−/− mice also demonstrate a more neutral SC pH than do their normal littermates, and their initial recovery from barrier perturbation is delayed. These findings confirm that sPLA2 enzymes perform important roles in epidermal development, and suggest that the sPLA2IIF isoform may be central to SC acidification and barrier function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.