- Retinoic acid ameliorates photoaged skin through RAR‑mediated pathway in mice
- UV irradiation
- Postirradiation treatment
- Histological examination
- Determination of collagen content
- Western blot analysis
- Statistical analysis
- 9-cis-13,14-Dihydroretinoic Acid Is an Endogenous Retinoid Acting as RXR Ligand in Mice
- 9-Cis-13,14-dihydroretinoic acid, a new endogenous mammalian ligand of retinoid X receptor and the active ligand of a potential new vitamin A category: vitamin A5
- Known RXR ligands and their physiological relevance
- Assessment of Etiologic Agents in Acne Pathogenesis
Retinoic acid ameliorates photoaged skin through RAR‑mediated pathway in mice
Photoaging refers to premature skin aging caused bychronic exposure to ultraviolet (UV) irradiation, especially theUVA (315–400 nm) and UVB (280–315 nm) components, which areregarded as the main cause of skin damage (1,2).
Photoaged skin is characterized by coarse wrinkles, dryness,laxity, dyspigmentation, telangiectasia, a leathery appearance andhistological changes, including variable epidermal thickness, solarelastosis and disorganization of collagen fibers (1,3).
UV irradiation induces the expression oftranscription factor activator protein-1 (AP-1), which plays animportant role in the mechanism of photoaging (4). AP-1, mainly composed of c-Jun andc-Fos proteins, increases matrix metalloproteinases (MMPs)transcription and decreases procollagen synthesis (3).
MMPs are a large family ofzinc-containing endopeptidases that are responsible for thedegradation of collagen and other extracellular matrix (ECM)proteins (5).
Among them, MMP-1(interstitial collagenase) initiates the cleavage of types I andIII fibrillar collagen in human skin, while MMP-3 (stromelysin-1)activates proMMP-1 and further degrades the collagen fragments(1,6). Rodents lack the MMP-1 gene, which isfunctionally replaced by the MMP-13 (collagenase-3) gene (5,7).
Therefore, MMP-3 and MMP-13 are the primary UV-inducedcollagenolytic enzymes in mouse skin. Type I procollagen issynthesized by dermal fibroblasts and subsequently converted intotype I collagen, which is the major structural protein in dermalECM.
In the process of photoaging, UV irradiation decreases type Iprocollagen synthesis, resulting in the loss of collagen content(3). the underlyingmechanism of photoaging, the regulation of MMPs and type Iprocollagen may be an effective strategy for the prevention andtreatment of photoaging.
Retinoic acid (RA), the bioactive metabolite ofvitamin A, plays a key role in regulating proliferation anddifferentiation of cutaneous cells (8). It has been widely used in thetreatment of dermatological disorders, such as acne, psoriasis,skin carcinoma and photoaging (9–12).
In particular, all-trans retinoic acid (ATRA) is considered thegold standard to treat photoaged skin (13). Topical ATRA could improve severalclinical and histological signs of photoaged skin, includingimproved skin appearance, increased anchoring fibrils and increaseddermal collagen (8,12–14).
Classically, the actions of RA are mediated by retinoid nuclearreceptors, retinoic acid receptor (RAR) and retinoid X receptor(RXR) (15). However, whether RAexerts its therapeutic effects on photoaged skin through retinoidnuclear receptors has not yet been elucidated.
Therefore, thepresent study aimed to explore whether the therapeutic effects ofRA on photoaged skin are mediated by RAR and/or RXR in mice and toinvestigate the underlying mechanism by histological examination ofcollagen fibers, determination of collagen content and detection ofMMP-3, MMP-13, type I procollagen, c-Jun and c-Fos proteinexpression in mouse skin.
Male ICR mice (8 weeks old) were obtained from theLaboratory Animal Center of Xi'an Jiaotong University (Xi'an,China). The animals were housed under controlled temperature(23±2°C), humidity (55±5%) and light (12-h light/dark cycle) withfree access to standard diet and water.
After acclimatization for 1week, 72 mice were randomly allocated into two groups:non-irradiated group (n=8) and UV-irradiated group (n=64). Thedorsal skin of mice (2×3 cm2) was shaved using anelectric razor, and this operation was repeated before UVirradiation and postirradiation treatment.
All animal experimentswere approved by the Laboratory Animal Administration Committee ofXi'an Jiaotong University and performed according to the Guidelinesfor Animal Experimentation of Xi'an Jiaotong University and theGuide for the Care and Use of Laboratory Animals published by theUS National Institutes of Health (NIH publication no. 85–23,revised 2011).
UV irradiation of mice was performed using the UVlight source provided by 2 UVA lamps (315–400 nm, peak wavelength:365 nm) and 4 UVB lamps (280–315 nm, peak wavelength: 312 nm) (bothfrom Beijing Lighting Research Institute, Beijing, China). Thedistance from the lamps to the animals' backs was 30 cm.
Theminimal erythema dose (MED) was preliminarily measured with a UVmeter (Lutron UV-340A, Taipei, Taiwan), and 1,200 mJ/cm2of UVA and 180 mJ/cm2 of UVB were assembled 1 MED inthis study. Mice were irradiated 3 times a week (Monday, Wednesdayand Friday) for 12 weeks. The irradiation dose was increased weeklyby 1 MED from 1 MED up to 4 MED and then maintained at 4 MED forthe rest weeks.
The non-irradiated group was treated identicallywith the lamps power off.
After 12 weeks of UV irradiation, mice in theUV-irradiated group were randomly reallocated into eight groupswith 8 mice per group: UV-irradiated plus vehicle (ethanol:propylene glycol, 7:3 v/v)-treated group (UV control group),UV-irradiated plus ATRA (Sigma-Aldrich, St.
Louis, MO, USA)-treatedgroup (ATRA group), UV-irradiated plus ATRA and AGN193109 (SantaCruz Biotechnology, Inc.
, Santa Cruz, CA, USA)-treated group (ATRA+ RAR antagonist group), UV-irradiated plus ATRA and HX531 (TocrisBioscience, Bristol, UK)-treated group (ATRA + RXR antagonistgroup), UV-irradiated plus TTNPB (Sigma-Aldrich)-treated group (RARagonist group), UV-irradiated plus TTNPB and AGN193109-treatedgroup (RAR agonist + RAR antagonist group), UV-irradiated plusSR11237 (Sigma-Aldrich)-treated group (RXR agonist group) andUV-irradiated plus SR11237 and HX531-treated group (RXR agonist +RXR antagonist group). ATRA and retinoid receptor-specific agonistsand antagonists were applied topically 5 times a week in 100 µlvehicle per treatment for 8 weeks. According to previous studies(16–19), these agonists and antagonists wereapplied at the following concentrations with slight modification:ATRA, 160 nM; TTNPB, 160 nM; AGN193109, 400 nM; SR11237, 160 nM;HX531, 400 nM. Eight mice in the non-irradiated group were used asthe normal group and treated with vehicle alone.
Twenty-four hour after the final treatment, micewere sacrificed by cervical dislocation under anesthesia, anddorsal skin was quickly removed.
The skin samples were fixed in 10%neutral buffered formalin for 24 h, embedded in paraffin andsectioned at 5 µm. Masson's trichrome staining was performed toexamine the skin collagen fibers.
The stained sections werecaptured under an Olympus BX51 light microscope equipped with aDP70 digital camera (Olympus, Tokyo, Japan).
Determination of collagen content
Hydroxyproline (Hyp) can be converted to theequivalent of collagen by multiplying the factor 7.46, consideringHyp is the almost exclusive amino acid of collagen and accounts for13.4±0.
24% of mammalian collagen in previous studies (20,21).
Hence, in this study, total Hyp content in the skin was measuredusing the Hyp assay kit (Nanjing Jiancheng BioengineeringInstitute, Nanjing, China) according to the manufacturer'sinstruction for the determination of collagen content.
Western blot analysis
Fresh skin samples were homogenized in ice-cold RIPAlysis buffer (Heart Biological Technology, Co., Ltd., Xi'an,China). Lysates were centrifuged at 14,000 × g for 10 min at 4°C,and the supernatants were collected as the total proteins.
Proteinconcentration was measured using the BCA protein assay kit(Beyotime Institute of Biotechnology, Shanghai, China). Each samplewas subsequently denatured by boiling in Laemmli loading buffer for5 min.
Equal amounts of protein were separated by 8–10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andtransferred to PVDF membranes (Millipore, Billerica, MA, USA).
After blocking with 5% non-fat dried milk for 2 h at 37°C, themembranes were incubated overnight at 4°C with the followingprimary antibodies: rabbit anti-MMP-3 monoclonal antibody (Cat. no.ab52915; 1:1,000 dilution), rabbit anti-MMP-13 polyclonal antibody(Cat. no.
ab39012; 1:1,000 dilution) (both from Abcam, Cambridge,MA, USA), rabbit anti-c-Jun monoclonal antibody (Cat. no. 9165;1:1,000 dilution), rabbit anti-c-Fos monoclonal antibody (Cat. no.2250; 1:1,000 dilution) (both from Cell Signaling Technology, Inc.,Danvers, MA, USA), goat anti-type I procollagen polyclonal antibody(Cat. no.
sc-8787; 1:500 dilution) and mouse anti-β-actinmonoclonal antibody (Cat. no. sc-47,778; 1:1,000 dilution) (bothfrom Santa Cruz Biotechnology, Inc.). After washing 3 times, themembranes were incubated with the appropriate horseradishperoxidase (HRP)-conjugated secondary antibodies for 1 h at 37°C,followed by enhanced chemiluminescence (Millipore). The signalswere captured, and the intensity of the protein bands wasquantified using Image J software (NIH, Bethesda, MD, USA)(22).
All data are presented as the means ± SD.Statistical analysis was performed using SPSS 19.0 software (SPSS,Inc., Chicago, IL, USA). Data were analyzed using one-way ANOVA ora Student's t-test to perform comparisons between two groups. AP-value of
9-cis-13,14-Dihydroretinoic Acid Is an Endogenous Retinoid Acting as RXR Ligand in Mice
The retinoid X receptors (RXRs) are ligand-activated transcription factors which heterodimerize with a number of nuclear hormone receptors, thereby controlling a variety of (patho)-physiological processes.
Although synthetic RXR ligands are developed for the treatment of various diseases, endogenous ligand(s) for these receptors have not been conclusively identified. We show here that mice lacking cellular retinol binding protein (Rbp1-/-) display memory deficits reflecting compromised RXR signaling.
Using HPLC-MS and chemical synthesis we identified in Rbp1-/- mice reduced levels of 9-cis-13,14-dihydroretinoic acid (9CDHRA), which acts as an RXR ligand since it binds and transactivates RXR in various assays.
9CDHRA rescues the Rbp1-/- phenotype similarly to a synthetic RXR ligand and displays similar transcriptional activity in cultured human dendritic cells. High endogenous levels of 9CDHRA in mice indicate physiological relevance of these data and that 9CDHRA acts as an endogenous RXR ligand.
Daily nutrition, in addition to being a source of energy, contains micronutrients, a class of nutrients including vitamins which are essential for life and which act by orchestrating a vast number of developmental and physiological processes.
During metabolism, micronutrients are frequently transformed into their bioactive forms. Nuclear hormone receptors are a family of proteins functioning as ligand-regulated transcription factors which can sense such bioactive molecules and translate those signals into transcriptional, adaptive responses.
Retinoid X receptors occupy a central place in this signaling as they directly interact, and thereby control, activities of several nuclear hormone receptors.
We report here the identification of a novel bioactive form of vitamin A, which is the first endogenous form of this vitamin capable to bind and activate retinoid X receptors.
Accordingly, we show that this single molecule displays biological activity similar to synthetic agonists of retinoid X receptors and coordinates transcriptional activities of several nuclear receptor signaling pathways. Those findings may have immediate biomedical implications, as retinoid X receptors are implicated in the control of a number of physiological functions and their pathology.
Citation: Rühl R, Krzyżosiak A, Niewiadomska-Cimicka A, Rochel N, Szeles L, Vaz B, et al. (2015) 9-cis-13,14-Dihydroretinoic Acid Is an Endogenous Retinoid Acting as RXR Ligand in Mice. PLoS Genet 11(6): e1005213. https://doi.org/10.1371/journal.pgen.1005213
Editor: Peter McCaffery, University of Aberdeen, UNITED STATES
Received: October 24, 2014; Accepted: April 13, 2015; Published: June 1, 2015
Copyright: © 2015 Rühl et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files. Transcriptomic data were deposited into the Gene Expression Omnibus database under accession no. GSE48573.
Funding: AK received a PhD fellowship from French Minister of Science, ANC and MWS were supported by the Agence Nationale de la Recherche and Fondation pour la Recherche Médicale, respectively. NR acknowledges the ANR (ANR- 11-BSV8-023) and the French Infrastructure for Integrated Structural Biology (FRISBI).
The work was also supported by TÁMOP-4.2.2.A-11/1/KONV-2012-0023 “VÉD-ELEM” project and by the TÁMOP-4.2.2.A-11/1/KONV-2012-0031 project. The project is implemented through the New Hungary Development Plan co-financed by the European Social Fund and the European Regional Development Fund.
Work at ARdL laboratory was supported by grants from the Spanish MINECO (SAF2010-17935), Xunta de Galicia (Grant 08CSA052383PR from DXI+D+i; Consolidación 2006/15 from DXPCTSUG; INBIOMED-FEDER “Unha maneira de facer Europa”; Parga Pondal Contract to BV).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: RR is owner of Paprika Bioanalytics BT; RR and MS are employees of Paprika Bioanalytics BT; WK, RR and ARdL are inventors on the patent for synthesis and biomedical applications of 9CDHRA.
Micronutrients such as vitamin A and polyunsaturated fatty acids are essential ingredients of mammalian diet and can act as bioactive molecules. Nuclear hormone receptors sense such molecular signals and accordingly regulate gene expression, thus functioning as ligand-controlled transcription factors.
Retinoid X receptors (RXRs) occupy a central place in nuclear receptor signaling as obligatory heterodimerization partners for several of those receptors.
RXR ligands can regulate the activity of only some heterodimers including for example LXR-RXR or PPAR-RXR, collectively called permissive heterodimers in opposition to non-permissive heterodimers, RAR-RXR, which cannot be activated by RXR ligands alone [1,2]. Ligand-dependent modulation might be particularly relevant for the control of a wide range of physiological events.
For example, among complex functions, working memory was shown sensitive to RXR ligand activities in mice , whereas at the cellular and molecular level, differentiation of monocyte-derived dendritic cells is one of the well characterized experimental models used to study the activities of RXR ligands [4,5].
Such ligands are also known to act as powerful inducers of apoptosis in cancer cells [6,7] or as modulators of lipid and glucose metabolism, which has stimulated their clinical development for the treatment of cancer and metabolic diseases . Recent studies on antidepressant or neuro-regenerative activities of RXR specific agonists suggest also their utility for the treatment of some neuropsychiatric or neurodegenerative disorders [3,9,10].
In parallel to development and use of synthetic RXR ligands, several endogenous agonists have been proposed [11,12,13], but their physiological relevance remains questionable for different reasons. For example, 9-cis-retinoic acid (9CRA), an isomer of all-trans-retinoic acid (ATRA), was proposed and largely accepted as an endogenous RXR ligand [14,15].
However, although 9CRA can bind and activate RXRs at low concentrations, it was either undetectable [16,17,18,19,20,21] or was not present in sufficient concentrations  to enable RXR-mediated signaling in mammalian organisms.
Docosahexaenoic acid (DHA), an alternative RXR ligand, was shown to bind and transactivate RXRs under pharmacological conditions [11,23,24], but in the physiological setting it can be detected in the brain mainly in esterified form contributing to e.g.
structural components of the cell, while the pool of this fatty acid available for RXR activation remains too low [25,26]. Finally, phytanic acid [27,28] also suggested to bind RXR was not conclusively proven to be physiologically relevant.
In this study, we addressed the nature of known and novel endogenous retinoids and their role in RXR signaling in vivo by chemical, molecular and functional studies in distinct models.
In order to search for endogenous retinoids which may act as RXR ligand(s), we first employed behavioral and pharmacological analyses sensitive to RXR signaling as a tool to identify animal models with reduced RXR signaling.
In particular, we focused on spatial working memory previously reported as dependent on RXR and not RAR functions and more importantly also dependent on RXR ligand activities .
Using delayed non-match to place (DNMTP) task, we found that mice carrying a null mutation of cellular retinol binding protein I (RBP1), known for its role in retinoid metabolism , display memory deficits which phenocopy the effect of the loss of function of Rxrγ, a functionally predominant RXR in control of working memory (Fig 1A and ref. ).
In particular, Rbp1-/- and Rxrγ -/- mice performed significantly worse when compared to wild type (WT) mice at 3 or 6 min inter-trial intervals (ITI) in DNMTP task, attaining chance level (complete forgetting) already at 6 min, whereas WT mice performed at chance level only at 12 or 18 min depending on individual (Fig 1A, see grey part of the left panel). These data suggest that RXR signaling is compromised in Rbp1-/- mice.
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Fig 1. Compromised RXR signaling in Rbp1-/- mice.
(a) Rbp1-/- (n = 8) and Rxrγ-/- (n = 7) mice acquired working memory DNMTP task similarly to WT (n = 8) mice (F[8,160] = 14, p
9-Cis-13,14-dihydroretinoic acid, a new endogenous mammalian ligand of retinoid X receptor and the active ligand of a potential new vitamin A category: vitamin A5
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The identity of the endogenous RXR ligand has not been conclusively determined, even though several compounds of natural origin, including retinoids and fatty acids, have been postulated to fulfill this role. Filling this gap, 9-cis-13,14-dihydroretinoic acid (9CDHRA) was identified as an endogenous RXR ligand in mice. This review examines the physiological relevance of various potential endogenous RXR ligands, especially 9CDHRA. The elusive steps in the metabolic synthesis of 9CDHRA, as well as the nutritional/nutrimetabolic origin of 9CDHRA, are also explored, along with the suitability of the ligand to be the representative member of a novel vitamin A class (vitamin A5).
Various studies have previously claimed the characterization of endogenous ligands of retinoid X receptor (RXR) as having the structure of either a retinoid or a fatty acid.1–5 Dawson and Xia6 summarized several studies on RXR ligands.
In all cases, however, the endogenous and nutrition-dependent levels required for RXR activation were virtually unknown, as they were either below the limit of detection or the amounts were too low for receptor activation.
3,7–9 The first endogenous, physiologically relevant RXR ligand, 9-cis-13,14-dihydroretinoic acid (9CDHRA), was recently characterized and found to induce moderate transcriptional RXR activation (≈ 10−6 to 10−7 M range).
10 This review will focus on the metabolic and nutrimetabolic pathways for the endogenous synthesis of 9CDHRA from related 13,14-dihydro retinoids, 9-cis retinoids, and 9-cis-13,14-dihydro retinoids and will examine potential nutritional precursors of 9CDHRA.
The metabolic pathway starting from these previously mentioned substances may support the notion that these substances represent a novel fully or partly independent pathway for vitamin A activity. It is proposed here that 9CDHRA may represent an end product of a novel vitamin A pathway for activation of the nuclear hormone receptors, the retinoic acid receptors (RARs), and, most importantly, the RXRs.
Known RXR ligands and their physiological relevance
Various derivatives, ranging from retinoids to fatty acids, have been proposed to function as endogenous ligands for RXR.
1–5 In particular, the potential endogenously occurring retinoid 9-cis retinoic acid was the subject of intense scrutiny by several research groups and became widely accepted as the endogenous RXR ligand.
11–16 Other groups with expertise in retinoid analysis, however, failed to detect 9-cis retinoic acid in humans and other mammals,7–10,14,17–21 thus calling into question the endogenous occurrence of this compound (Figure 1A7,10,14,17).
It seems that 9-cis retinoic acid may be found endogenously only after administration of high doses of synthetic retinoids or after intake of high amounts of foods that are rich in vitamin A derivatives retinol and retinyl esters, ie, amounts reached only in nutritional intervention studies.22,23
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(A) Chromatogram of retinoic acid isomers in mouse brain and mouse serum, obtained by high-performance liquid chromatography (HPLC)–UV (adapted from Rühl et al10,); (B) Internal probe of a 9CRA-coeluting peak found by Kane et al14,in mouse pancreas samples with chromatograms of chemical standards of 9CRA and ATRA in UV detection mode (right side) and tandem mass spectrometry (MS/MS) detection mode (left side with MS/MS settings of 301–205 m/z). Methodology: Pancreas samples from mice were obtained from Dr W. Krężel and were analyzed by the methods described previously,7 with modifications outlined by Rühl et al.10 In this setup, no additional prefilter was used before the HPLC column. This resulted in shorter retention times, as seen for ATRA when compared with Figure 1A; (C) 9-Cis isomerization pathway from ATRA to 9CRA, 13CRA and 9,13DCRA as well as from ATDHRA to 9CDHRA; (D) UV-induced isomerization of ATRA by electromagnetic radiation in the visible range (adapted from Kane et al17), with arrows marking individual isomers of retinoic acid.Abbreviations: ATDHRA, all-trans-13,14-dihydroretinoic acid; ATRA, all-trans retinoic acid; 9CDHRA, 9-cis-13,14-dihydroretinoic acid; 9CRA, 9-cis retinoic acid; hv, used here as a representative term indicating, in this reference, electromagnetic radiation; 13CRA, 13-cis retinoic acid; 9,13DCRA, 9,13-dicis retinoic acid.
There are several technical issues that may have led to some confusion in the detection of endogenous 9-cis retinoic acid. The first claim for the identification of 9-cis retinoic acid as an RXR ligand was made by Heyman et al1 and was the analysis of high-performance liquid chromatography (HPLC) profiles with UV detection using coelution alone as an indicator.
9-Cis retinoic acid was identified and claimed to be endogenously present. Unfortunately, this method of identification and detection was subsequently proven to be not entirely specific and not conclusive enough because various derivatives may also coelute with 9-cis retinoic acid.
7,10,14 More-conclusive evidence of the presence of a given compound than HPLC coelution would require the UV spectrum of the substance to be identified along with a complete mass spectrum or, better still, selective highly specific mass fragmentation patterns of the derivative to be identified.
7,10,24,25 Indeed, the identification of 9-cis retinoic acid by HPLC coelution alone should be considered as a tentative confirmation of low security, while strong evidence of the endogenous existence of 9-cis retinoic acid is still lacking (reviewed by de Lera et al26).
These studies into whether 9-cis retinoic acid is an endogenous derivative were performed more than 20 years ago, and developments in analytical techniques, especially HPLC coupled with mass spectrometry (MS), have improved significantly, particularly with regard to selectivity and sensitivity.
In recent years, 2 groups have established highly sensitive HPLC–MS methodologies for retinoid analysis in the low range of 0.01 to 0.001 ng/ml (or ng/g) for the quantification of various retinoids, including retinoic acid isomers such as 9-cis retinoic acid.
20,27 In one case a peak was identified using tandem mass spectrometry (MS/MS) under conditions that were specific for retinoic acids (301–205 m/z, using HPLC–MS/MS), and a peak detected in a pancreas sample, which coeluted with 9-cis retinoic acid, and endogenous concentration values were calculated to be in the range of 4 to 6 ng/g.
14 It was claimed that “these data validate 9CRA [9-cis retinoic acid] as a naturally occurring metabolite of vitamin A.”14 However, parallel UV detection of this peak was not performed, and it was argued in a subsequent review that the use of parallel UV detection is limited for physiological measurements.
12 This statement is unfortunately incorrect, and the use of UV detection, is indeed feasible, as demonstrated in various other studies.7,8,23,28,29 Concentrations of around 1 ng/ml (or ng/g), ie, lower than the claimed endogenous levels of 4 to 6 ng/g (0.1–0.2 nM) in pancreas,14 can easily be detected by UV measurements.
7,8,28 Confirmation of these results was attempted using our analytical methodology and mouse pancreas samples (Figure 1B). In this case, a peak was indeed detected in the MS/MS track (9.2 min retention time in MS/MS mode) and with a better separation in the UV track (9.3 min retention time in UV chromatogram).
However, this substance only coelutes closely to 9-cis retinoic acid (9.0 min retention time in MS/MS mode and 9.1 min in the UV chromatogram), and it is obvious, with this analytical setup, that this peak has a slightly longer retention time than the 9-cis retinoic acid standard (Figure 1B).
Our group is currently focused on the identification of this retinoid, and therefore additional data for peak identification, such as a UV spectrum to identify peaks, cannot be shown yet.
On the basis of the data reported by Kane et al,14 it is doubtful that 9-cis retinoic acid is an endogenously occurring retinoid in pancreas.
Once again, it should be noted that a simple coelution without additional structural elucidation is insufficient to claim definitively the existence of endogenous 9-cis retinoic acid.
The appearance of a peak with specific MS/MS characteristics coeluting with 9-cis retinoic acid has already been confirmed in a study by Rühl,7 and it was proven by parallel UV detection that this peak did not correspond to 9-cis retinoic acid.
In a second ultrasensitive retinoid analysis, the presence of potentially endogenous 9-cis retinoic acid was also determined using highly sensitive MS/MS detection.27 This identification of 9-cis retinoic acid was further proven by coelution with standards, and endogenous levels of 0.027 ng/ml (
Assessment of Etiologic Agents in Acne Pathogenesis
Retinoids, glucocorticoids and thyroid hormone, bind to cytoplasmic receptors inducing signals, which in the presence of a ligand, are transmitted to the nucleus. At this location, these response elements bind to specific DNA sequence motifs, thereby affecting transcription.
Two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are present in skin. Sebocyte activity and differentiation in vitro depends on vitamin A and synthetic retinoids.
Accordingly, sebocytes in vitro express copies of several retinoid factors such as RAR and RXR , whose equilibrium interaction with coactivator and corepressor proteins is altered by binding to retinoids, thus altering transcription of several proteins.
Retinoids down-regulate lipid synthesis, decrease late-stage sebocyte differentiation, and inhibit sebocyte proliferation. Thus far, the most potent retinoid in inhibiting proliferation and differentiation is isotretinoin.
Retinoid receptors are expressed in a site-specific manner in the skin.
RARs have inverse agonists that repress basal transcription, neutral antagonists that do not change transcription but block transcriptional activation by agonists or repression by inverse agonists, and agonists. In keratinocytes both RAR agonists and inverse agonists regulate markers of abnormal differentiation and inflammation.
Retinoic acid also affects the infundibulum. Isotretinoin, adapalene, tazarotene, and other retinoids (also anti-inflammatory) are comedolytic and anticomedogenic.
One µmol 13-cis-retinoic acid decreases the rate of cell division in the infundibulum in vitro, which is possibly its major therapeutic effect.
 Furthermore, infundibular keratinocytes undergo parakeratosis, instead of the normal differentiation, when exposed to 1 µmol 13-cis retinoic acid.
Isotretinoin also interferes with neutrophilic granulocyte and monocyte function. Migration, phagocytosis, superoxide generation, myeloperoxidase-dependent killing function, and antioxidant effects are disrupted by isotretinoin. Isotretinoin also increases hydroxyl radical formation by zymosan stimulated neutrophiles.
Isotretinoin does not bind cellular retinoic acid binding protein-II or nuclear receptors. Isotretinoin/retinol combinations have comparable antiproliferative effects on sebocytes to isotretinoin alone. Triglycerides are lowered more with isotretinoin treatments alone than with combination or retinol alone treatments.
Isotretinoin/retinol combinations lowered triglycerides more than retinol alone. It seems retinol may be a partial agonist of isotretinoin's activity in lowering triglycerides. Under isotretinoin treatment, isotretinoin maintains a relatively constant intracellular concentration (60 nmol/L) inside of the sebocytes.
 Tretinoin, in contrast, rises 5 times (to 500 nmol/L) under isotretinoin treatment. Under retinol treatment sebocytes' retinol concentration increases to a constant 160 nmol/L and the concentration of retinyl palmitate doubles.
 Under the combination therapy retinoic acid levels rise to the same level as in treatment with retinoic acid alone, but the levels of retinol and retinyl palmate rise higher than under treatment with retinol alone. The inhibitory effect of isotretinoin is not affected by the presence of retinol.
Isotretinoin's effects could be mediated by the constant low concentration of isotretinoin or the elevated tretinoin concentrations in sebocytes after isotretinoin treatment.
In preclinical models oral 13-cis retinoic acid, 9-cis retinoic acid, and all-trans retinoic acid have the same activity. Additionally,13-cis retinoic acid decreases sebum excretion the most, followed by 9-cis retinoic acid and alltrans retinoic acid.
Only 13-cis retinoic acid shows sebosuppression. Interestingly, 13-cis retinoic acid does not bind nuclear receptors.
 Thus, either sebosuppression is not nuclear receptor mediated or 13-cis retinoic acid is first metabolized to a receptor-binder at the level of the sebaceous gland.
Retinol, retinal, and all-trans retinoic acid are inactivated by P-450 isozymes to 4-hydroxy-compounds, and 4-oxo-retinoic acid to more polar metabolites.
 One allele of P-4501A, one of the most active P-450 isozymes that can metabolize retinoids, is overexpressed in acne patients.
This isozyme may rapidly metabolize the endogenous retinoids causing a deficit of active natural retinoids, which may aid in acne causation.
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