Elastin Promoter Activity Research Articles

A blackberry-dill extract combination synergistically increases skin elasticity.

The loss of structural elastin due to intrinsic and extrinsic ageing results in the skin's inability to stretch and recoil (decrease in elasticity) and manifests as loss of skin firmness and sagging. While other extracellular matrix (ECM) components such as collagen and hyaluronic acid are continually synthesized and assembled through life, elastic fibres are not. Elastic fibre assembly and functionality require fibre cross-linking, induced by the lysyl oxidase-like (LOXL) enzymes, which sharply decrease during ageing. To evaluate the enhanced elastogenic effect of a blackberry-dill extract combination, which was hypothesized to induce elastin fibre component synthesis, fibre cross-linking and reduce elastin fibre degradation. The blackberry and the dill extracts were tested separately and in combination to confirm single ingredient bioactivity and synergistic benefits. Human skin explants, dermal fibroblasts, elastase assays, ELISAs, quantitative real-time PCRs and spectrofluorometer measurements were used. Moreover, a double-blinded, placebo-controlled clinical study was carried out to assess skin elasticity using Cutometer and histologically from biopsies. The blackberry extract induced elastin gene expression, elastin promoter activity and inhibited elastic fibre degradation by matrix metalloproteinases (MMPs) 9 and 12. The dill extract induced elastin, collagen and LOXL1 gene expression, resulting in enhanced fibre cross-linking in human skin explants. Clinically, the blackberry and dill combination treatment displayed synergistic pro-elasticity activity as compared to each ingredient alone and placebo. Taken together, these results demonstrated the two multimodal plant-based extracts complemented each other in terms of bioactivity and resulted in a synergistic elastogenesis induction.

Extracts from Glycine max (soybean) induce elastin synthesis and inhibit elastase activity

Elastic fibres are essential extracellular matrix components of the skin, contributing to its resilience and elasticity. In the course of skin ageing, elastin synthesis is reduced, and elastase activity is accelerated, resulting in skin sagging and reduced skin elasticity. Our studies show that non-denatured Glycine max (soybean) extracts induced elastin promoter activity, inhibited elastase activity and protected elastic fibres from degradation by exogenous elastases in vitro. Mouse and swine skins topically treated with soybean extracts showed enhanced elastic fibre network and increased desmosine content. Elastin expression was also augmented in human skin transplanted onto SCID mice in response to soy treatment. These data suggest that non-denatured soybean extracts may be used as skin care agents to reduce the signs of skin ageing.

Skin elastic fibres: regulation of human elastin promoter activity in transgenic mice.

Elastic fibres form an extracellular network which provides elasticity and resilience to tissues such as the skin. To study the regulation of human elastin gene expression, we have developed a line of transgenic mice which harbour 5.2 kb of human elastin gene promoter region in their genome. This promoter is linked to the chloramphenicol acetyltransferase (CAT) reporter gene which allows determination of the expression of human elastin promoter in different tissues. The highest CAT activity was found in the lungs and aorta, tissues rich in elastin, while lower levels were detected in a variety of other tissues, including skin. Assay of CAT activity in the lungs of fetal and newborn animals revealed high activity which progressively declined during the postnatal period up to six months. Thus, there was evidence of tissue-specific and developmentally regulated expression of the human elastin promoter activity in these mice. These animals were then used to examine the expression of the elastin gene by a variety of factors which have previously shown to alter elastin gene expression, as determined at the mRNA or protein levels. First, injection of transforming growth factor beta 1 (100 ng) subcutaneously into the transgenic animals resulted in a time-dependent elevation of the promoter activity up to 10-fold after a single injection. Secondly, enhancement of the human elastin promoter activity by interleukin 1 beta injected subcutaneously resulted in an approximately 10-fold elevation of the CAT activity. Finally, subcutaneous injection of these animals with triamcinolone acetonide or dexamethasone, two glucocorticosteroids in clinical use, resulted in marked enhancement of human elastin promoter activity. Similar changes were noted in fibroblast cultures established from the transgenic animals. These data indicate that the 5.2 kb upstream segment of the human elastin gene contains cis-elements which allow tissue-specific and developmentally regulated expression of the human elastin promoter. Furthermore, this segment of the gene contains responsive elements to a variety of cytokines and pharmacological agents. Collectively, these data indicate that elastin gene expression in the skin in vivo can be regulated at the transcriptional level.

Regulation of elastin promoter by lysyl oxidase and growth factors: Cross control of lysyl oxidase on TGF-β1 effects

Lysyl oxidase (LOX) plays a key role in the maturation of the extra-cellular matrix, by inducing the formation of lysyl cross-links in collagen and elastin molecules. Beside its enzymic activity, LOX is able to regulate the promoter of collagen III, one of its natural substrates. In this paper we demonstrated that LOX regulates also the promoter of elastin, inducing an important activation of its activity. In order to define the pathways used by LOX to achieve its effect, we activated some of the main fibrogenic signal pathways and studied the consequences on LOX effects on the promoter. TGF-β1 activated most of the elastin promoter constructs that we studied, except for an inhibitory region contained in the region between − 1500 and − 1000 bp. The treatment with TGF-β1 abolished completely the activation induced by LOX. LOX-over-expression coupled with TGF treatment abolished both effects in the − 500 bp region. The treatment with CTGF also inhibited LOX effect, although to a lesser extent. However, CTGF behaved quite differently from TGF-β1 suggesting that it is not necessarily the mediator of TGF effects. Basic FGF, the other fibrogenic factor that we tested, again abolished LOX-dependent activation, but by itself did not affect elastin promoter activity. Because TGF-β1 activating effects, we used EMSA to examine the transcription factor binding patterns in presence of LOX, TGF-β1 or both. The study showed that LOX reverted the patterns of several DNA–protein complexes along the 1.5 kb of the studied promoter region. Most were affected by both LOX and TGF-β1, while on some only TGF-β1 was effective. LOX presence mostly inhibited the TGF-regulated complexes. Many of those included SMAD transcription factors. Two more restricted regions binding AP1 and SMAD were identified as mediators of LOX effects and of LOX and TGF-β1 cross-inhibition.

A method for rapid evaluation of photoaging by measuring fluorescence intensity of green fluorescent protein due to elastin promoter activity

Summary Background Photoaging increases the deep furrows of the facial skin. This can be seen histologically as solar elastosis in the upper layer of the dermis. In sunlight, ultraviolet B (UVB) light plays a pivotal role in forming the wrinkles, because it enhances elastin gene expression and matrix metalloproteases (MMPs) at the transcriptional level in dermal fibroblasts. Sunscreens are believed to inhibit UVB-induced wrinkle formation in humans, but few methods have accurately assessed the protective effects of sunscreens in vivo to date. A major reason is that it takes a long time to cause the deep facial lines. Objective To develop a rapid and simple in vivo evaluation system, to assess the inhibitory effects of sunscreens against photoaging, a new experimental method was studied. Methods The promoter region of the human elastin gene was cloned into a green fluorescent protein (GFP) vector. The plasmid DNA was transfected into 3T3 cells and stable transfectants (3T3/Eln-GFP) were established. These cells were injected into the dorsal skin of nude mice. After UVB irradiation, the GFP fluorescence intensity was measured by fluorescence emission spectroscopy (FEMS) on the dorsal skin of the mouse. Results UVB irradiation of 3T3/Eln-GFP cells increased both the fluorescence intensity and the expression of GFP. Increased GFP fluorescence intensity could be measured from the body surface with FEMS at the injected site of the cells after UVB irradiation in vivo . Conclusion A rapid and simple evaluation system for human elastin promoter activity by UVB was established, and we propose it to be an useful method for objective assessment of sunscreens designed to prevent wrinkle formation.

Development of UV-induced squamous cell carcinomas is suppressed in the absence of SPARC.

SPARC (Secreted Protein Acidic and Rich in Cysteine) is a multifunctional glycoprotein belonging to a group of matrix-associated factors that mediate cell-extracellular matrix interactions but have no structural roles. In the present study we investigated the contribution of SPARC to factors that influence the development of skin tumors in response to UV irradiation. A hairless SPARC-null mouse was developed and compared to control SKH1 hairless mice in terms of skin tumor induction and extracellular matrix changes occurring in response to UV-irradiation. Following 23 weeks of exposure to UVB totaling 14.5 J per cm(2), tumor development in the wild-type mice was severe, with an average of over 20 tumors per mouse, many of which were squamous cell carcinomas. Conversely, the SPARC-null mice were strikingly tumor-resistant, developing no squamous cell carcinomas and averaging less than one small papilloma per mouse. SPARC was undetectable immunohistochemically in skin from the non-irradiated control group yet was present in relatively high quantities in the basal and superficial areas of the tumor mass. The SPARC-null mice also exhibited a limited contact hypersensitivity response and were refractory to UV induced immune suppression. In conclusion, SPARC appears to have a crucial role in mediating tumor formation in response to UV irradiation.

The polyhydroxy acid gluconolactone protects against ultraviolet radiation in an in vitro model of cutaneous photoaging.

Ultraviolet (UV) radiation damages skin through a variety of mechanisms, including the generation of free radicals. Gluconolactone is a polyhydroxy acid (PHA) that is capable of chelating metals and may also function by scavenging free radicals, thereby protecting skin from some of the damaging effects of UV radiation. This study measured the ability of gluconolactone to protect against UV radiation-induced damage. The ability of gluconolactone to prevent UV radiation-induced elastin promoter activation was determined in vitro using a transgenic model of cutaneous photoaging. Gluconolactone was also evaluated to determine its ability to promote the formation of sunburn cells in human skin after exposure to UV radiation. Gluconolactone provided up to 50% protection against UV radiation, as measured in our in vitro system, and did not significantly increase sunburn cells in human skin. These results demonstrate the ability of the PHA gluconolactone to protect against UV radiation-induced elastin promoter activation. In addition, in vivo studies demonstrated that gluconolactone treatment does not result in a significant increase in sunburn cells. Further investigation of this and other PHAs is necessary to identify their potential role in preventing and repairing cutaneous photodamage.

Transcriptional regulation of pulmonary elastin gene expression in elastase-induced injury.

Previously we have shown that treatment of confluent, pulmonary fibroblast cultures with elastase results in upregulation of elastin mRNA and protein levels. In the present study we focused on determining the level at which elastin expression is upregulated after elastase exposure. We examined as models for this investigation elastin gene expression in primary pulmonary fibroblast cells during the transition from subconfluent to confluent cultures and in confluent, matrix-laden cultures treated briefly with elastase. In addition, we extended our studies to mice that were given an intratracheal dose of elastase; the effects on lung elastin mRNA and elastin promoter activity levels were measured and compared with results from in vitro cell models. The results demonstrate that upregulation of elastin gene expression during the transition of subconfluent to confluent cultures and after elastase injury is associated with an increase in the level of transcription both in vitro and in vivo. Furthermore, intratracheal administration of elastase to transgenic mice illustrates that the increased levels of elastin mRNA are accompanied by increased activity of the elastin gene promoter in cells spatially positioned near major sites of tissue injury.

NF-kappaB induced by IL-1beta inhibits elastin transcription and myofibroblast phenotype.

Interleukin (IL)-1beta released after lung injury regulates the production of extracellular matrix components. We found that IL-1beta treatment reduced the rate of elastin gene transcription by 74% in neonatal rat lung fibroblasts. Deletion analysis of the rat elastin promoter detected a cis-acting element located at -118 to -102 bp that strongly bound Sp1 and Sp3 but not nuclear factor (NF)-kappaB. This element mediated IL-1beta-induced inhibition of the elastin promoter. IL-1beta treatment did not affect the level of Sp1 but did induce translocation of the p65 subunit of NF-kappaB. Overexpression of p65 decreased elastin promoter activity and markedly reduced elastin mRNA. Immunoprecipitation studies indicated an interaction between the p65 subunit and Sp1 protein. Microarray analysis of mRNA isolated after overexpression of p65 or treatment with IL-1beta revealed downregulation of alpha-smooth muscle actin and calponin mRNAs. Expression of these genes is associated with the myofibroblast phenotype. These results indicate that IL-1beta activates the nuclear localization of NF-kappaB that subsequently interacts with Sp1 to downregulate elastin transcription and expression of the myofibroblast phenotype.

Reactive oxygen species activate the human elastin promoter in a transgenic model of cutaneous photoaging.

Generation of free radicals has been shown to play a role in cutaneous alterations resulting from ultraviolet radiation. Cells from a previously described in vitro transgenic model of cutaneous photoaging were exposed to reactive oxygen species to determine if this results in elastin promoter activation. Reactive oxygen species were generated using a hypoxanthine and xanthine oxidase system, and elastin promoter activation was measured using cells derived from transgenic mice containing the human elastin promoter. Free radical generation resulted in a greater than sixfold increase in elastin promoter activity, and this increase was blocked with the addition of catalase. Elastin promoter activation may play a role in the generation of solar elastosis in photoaged skin. Utilizing hypoxanthine and xanthine oxidase with the in vitro transgenic photoaging model results in a sensitive system for evaluating agents that may prevent oxidative damage.

The nitroxide Tempol affords protection against ultraviolet radiation in a transgenic murine fibroblast culture model of cutaneous photoaging.

The generation of reactive oxygen species is among the various mechanisms by which ultraviolet radiation damages skin. Tempol, a superoxide dismutase analogue which readily penetrates cell membranes when administered exogenously, has been shown to provide protection against some forms of oxygen-dependent damage. In this study, we measured the ability of Tempol to protect against ultraviolet A- and ultraviolet B-induced damage, using a previously described transgenic mouse model of cutaneous photoaging. The ability of Tempol to prevent ultraviolet radiation-induced elastin promoter activation was determined in vitro. Tempol provided over 50% protection against ultraviolet B and over 70% protection against ultraviolet A as measured in our in vitro system. These results demonstrate the ability of the superoxide dismutase mimic, Tempol, to protect against ultraviolet induced elastin promoter activation. This compound could be a useful pharmacological agent to prevent cutaneous photoaging.

Basic Fibroblast Growth Factor Decreases Elastin Gene Transcription through an AP1/cAMP-response Element Hybrid Site in the Distal Promoter

Previous studies demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in pulmonary fibroblasts. In this study we pursue the identification of the element and the trans-acting factors responsible. Gel shift analyses show that bFGF increases protein binding to a sequence located at -564 to -558 base pairs (bp), which possesses homology to both AP1 and cAMP-response consensus elements yet displays a unique affinity for heterodimer binding. Site-directed mutation of the -564- to -558-bp sequence results in an increase in promoter activity and abrogates the effect of bFGF. Western blot analysis shows that bFGF induces a sustained increase in the steady-state levels of Fra 1, and co-transfection of a Fra 1 expression vector with an elastin promoter reporter construct results in an inhibition of elastin promoter activity. Overall the results suggest that bFGF represses elastin gene transcription by increasing the amount of the Fra 1 that subsequently binds to the -564- to -558-bp as a heterodimer with c-Jun to form an inhibitory complex. We propose that the identified bFGF response element can serve to down-regulate elastin transcription in elastogenic cells and, conversely, can serve to up-regulate elastogenesis in cells where endogenous bFGF signaling is attenuated or altered.

The role of NF-1 factors in regulation of elastin gene transcription.

The -195- to -500-bp region of the human elastin promoter has been shown to convey high activity in neonatal rat aortic smooth muscle cell and pulmonary fibroblast cell cultures. In addition, this region has been implicated in controlling the differential basal level of elastin transcription in these two cell types. The overall goal of this study was to define the positive element(s) within the -195- to - 500-bp region and to identify the trans-acting factors binding to this sequence. A combination of deletion and linker scan mutational analyses localizes the positive element between -401 and -415 bp. Gel shift analyses demonstrate that the positive element binds NF-1 family members. Co-transfection of a CTF1 expression vector in Drosophila Schneider cells shows the ability of an NF-1 family member to activate elastin promoter activity through this site. Comparative Western and Southwestern blot analyses of nuclear extracts isolated from SMC and lung fibroblasts lay the foundation for possible differential regulation of elastin transcriptional levels via cell specific expression of different NF-1 family members.

All- trans-retinoic acid down-regulates elastin promoter activity elevated by ultraviolet B irradiation in cultured skin fibroblasts

Topical tretinoin therapy produces clinical improvements in the fine wrinkling of photodamaged skin, possibly by enhancement of collagen synthesis. A major biochemically and histologically detectable change in photodamaged skin is the accumulation of abnormal elastic fibers (elastotic material). However, little is known about the effects of retinoic acid and ultraviolet B (UVB) on elastin gene expression. Consequently, we examined the effects of all- trans-retinoic acid (t-RA) and UVB on elastin gene expression in cultured human skin fibroblasts in vitro. Elastin mRNA gene expression was up-regulated in response to UVB by ≈3-fold, in a dose dependent manner, between 3 and 10 mJ/cm 2 doses. Similar results were obtained by chloramphenicol acetyltransferase assay, in which a maximal promoter activation more than 5.4-fold that in nonirradiated controls occurred after a single dose of 20 mJ/cm 2. Also, t-RA inhibited the increase in elastin mRNA level following a single exposure to UVB by approximately 16%, and the increase in promotor activity by about 65%. The inhibitory effect of t-RA on elastin induced by UVB was also demonstrated by indirect immunofluorescence studies. Taken together, t-RA down-regulated human elastin gene expression elevated by a single exposure to UVB at transcriptional and possibly protein levels. These results suggest that the anti-photoaging effect of t-RA may be related, at least in part, to down-regulation of elastin gene expression elevated by UVB.

Evaluation of sunscreens with various sun protection factors in a new transgenic mouse model of cutaneous photoaging that measures elastin promoter activation

Background: Long-term sun exposure can cause major alterations in the papillary dermis, resulting in the deposition of massive amounts of abnormal elastic material, termed solar elastosis. Previous work has demonstrated that this type of photodamage is accompanied by an increase in elastin and fibrillin messenger RNAs and elastin promoter activity. Objective: Our purpose was to develop a rapid and sensitive in vivo method for evaluating compounds offering protection against cutaneous photodamage. Methods: Using a line of transgenic mice that expresses the human elastin promoter linked to a chloramphenicol acetyltransferase reporter gene, we applied sunscreens with various sun protection factors to 5-day-old mice followed by 30 minimal erythema doses of solar simulating radiation for three consecutive days. Results: Solar simulating radiation alone resulted in a fivefold increase in elastin promoter activity. Sun protection factors of 2, 4, 8, and 15 yielded a reduction in promoter activity by 2.8%, 42.5%, 58.1%, and 70.3%, respectively. Conclusion: These results confirm the use of this system as a rapid and sensitive in vivo model for evaluating compounds that protect against photodamage. (J Am Acad Dermatol 1997;37:725-9.)

Identification of Novel Glucocorticoid-Response Elements in Human Elastin Promoter and Demonstration of Nucleotide Sequence Specificity of the Receptor Binding

Glucocorticoids exert their action on gene expression through activation of cytoplasmic glucocorticoid receptors (GRs) that bind to glucocorticoid response elements (GREs). The consensus GRE consists of two half sites (underlined), AGAACANNNTGTTCT. We have recently cloned the entire human elastin gene. Nucleotide sequencing of the promoter region disclosed the presence of three putative GREs with the downstream half-site sequence TGTTCC that has homology with the consensus GRE, although the upstream half site showed no homology. To examine the functionality of these putative GREs in binding to the GRs, we performed gel mobility shift and supershift assays with synthetic oligomers containing the putative GREs and a recombinant GR protein, expressed in a baculovirus system. All three GREs identified in the elastin promoter bound the receptor. A chimeric oligonucleotide containing the upstream consensus GRE half site and the downstream elastin promoter GRE half site was capable of binding the receptor, and this binding could be competed with the elastin promoter GRE. Nonconservative substitution of single nucleotides (positions 1-6) in the elastin GRE indicated that mutations in the positions 1-3 and 6 had relatively little effect, but substitutions in positions 4 and 5 rendered the oligomer less effective in competing for the binding. These observations suggest that the downstream half site of GREs in the human elastin promoter is sufficient for receptor binding and certain nucleotides are critical for the efficient binding. The results also imply that the three GREs within the human elastin promoter are active and mediate the glucocorticoid-induced up-regulation of human elastin promoter activity.

Developmental regulation of elastin gene expression.

Developmental regulation of elastin gene expression appears to be exerted primarily at the transcriptional level. Although the elastin gene promoter possesses features of "housekeeping" genes, these characteristics do not preclude transcriptional regulation as shown with a number of other gene promoters of this class. Direct evidence for transcriptional regulation has been obtained by nuclear run-on analysis of nuclei isolated from developing lung and aortic tissues and indirectly through elastin promoter activity in transgenic mice and transient tissue transfections of embryonic lung and aortic tissues. Although several different modulators have been proposed to control the developmental activation of elastin gene expression, only insulin-like growth factor I has been experimentally linked to increased transcription by in vivo studies. This link is specific for aortic smooth muscle cells in which cell cycle control appears intimately associated with elastogenesis. Recent studies suggest that progress in understanding developmental activation of the elastin gene lies in transgenic models and organ transfection assays that assess the direct relevancy of the modulators and cis- and trans-acting factors involved.

8-methoxypsoralen and ultraviolet a radiation activate the human elastin promoter in transgenic mice: in vivo and in vitro evidence for gene induction.

Treatment of skin diseases with the combination of 8-methoxypsoralen and ultraviolet A radiation (PUVA) results in clinical alterations in treated skin that resemble those observed in chronically photodamaged skin. The PUVA-treated patients develop nonmelanoma skin cancers, pigmentary alterations and wrinkling characteristic of sun-induced changes. The major alteration in the dermis of sun-damaged skin is the deposition of abnormal elastic fibers, termed solar elastosis. Up-regulation of elastin promoter activity in dermal fibroblasts explains the excess elastic tissue but not the reason for the aberrant morphology of the elastotic material. In order to study photoaging in an experimental system, we utilized a transgenic mouse line that expresses the human elastin promoter/chloramphenicol acetyltransferase construct in a tissue-specific and developmentally regulated manner. Although UVB radiation has been demonstrated to increase promoter activity in vitro, UVA fails to demonstrate a similar effect at the doses utilized. In this study, we demonstrate the ability of PUVA treatment to up-regulate elastin promoter activity both in vitro and in vivo. These data help to explain the development of photoaging in sun-protected PUVA-treated skin. We attribute the up-regulation of elastin promoter activity in response to PUVA to the formation of DNA photoadducts, which do not occur in response to UVA radiation alone.

Ultraviolet Radiation Activates the Human Elastin Promoter in Transgenic Mice: A Novel In Vivo and In Vitro Model Of Cutaneous Photoaging

The major alteration in photoaged skin is the deposition of massive amounts of abnormal elastic material, termed solar elastosis. In previous work, it has been shown that solar elastosis is accompanied by increased abundance of elastin and fibrillin mRNAs and upregulation of elastin promoter activity. Using a transgenic mouse line, which expresses the human elastin promoter, linked to a chloramphenicol acetyltransferase reporter gene, in a tissue-specific and developmentally regulated manner, we investigated the effects of ultraviolet A radiation and ultraviolet B radiation on human elastin promoter activity in vivo and in vitro. Irradiation of mice with a single dose of ultraviolet B radiation (491.4 mJ/cm2) resulted in an increase up to 8.5-fold in promoter activity, whereas a more modest increase of 1.8-fold was measured with ultraviolet A radiation (38.2 J/cm2). In addition, in vitro studies revealed over a thirtyfold increase in elastin promoter activity in response to ultraviolet B radiation (5.5 mJ/cm2), whereas no change was measured in response to ultraviolet A radiation (2.2 J/cm2). These results confirm the role of ultraviolet B radiation in elastin promoter activation in photoaging, and identify ultraviolet A radiation as a contributing factor. This system should serve as a useful in vivo and in vitro model to study cutaneous photoaging, and for testing compounds that may protect against cutaneous photodamage.

Glucocorticosteroids Up-Regulate Human Elastin Gene Promoter Activity in Transgenic Mice

Recent characterization of the human elastin gene identified three putative glucocorticoid responsive elements (GRE) within the 5'-flanking DNA. To test the functionality of these cis-elements, transgenic mice that express a human elastin promoter-reporter gene (CAT) construct in a tissue-specific manner were injected with triamcinolone acetonide (TMC) or dexamethasone (DEX), two glucocorticosteroids in clinical use. Subcutaneous injection of these glucocorticoids resulted in a marked, up to 28-fold, enhancement of the CAT activity in the skin at the site of injection. Similarly, intraperitoneal injection of DEX resulted in significant increases in the elastin promoter activity in various internal organs. Furthermore, incubation of skin fibroblast and aortic smooth muscle cell cultures established from the transgenic animals with TMC (10 ng/ml) resulted in marked increases in the elastin promoter activity. These studies demonstrate that glucocorticosteroids act as powerful up-regulators of human elastin promoter activity in transgenic mice.