MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

Molecular Interactions of Selective Agonists and Antagonists with the Retinoic Acid Receptor γ.

All-trans retinoic acid (ATRA), the major active metabolite of all-trans retinol (vitamin A), is a key hormonal signaling molecule. In the adult organism, ATRA has a widespread influence on processes that are crucial to the growth and differentiation of cells and, in turn, the acquisition of mature cell functions. Therefore, there is considerable potential in the use of retinoids to treat diseases. ATRA binds to the retinoic acid receptors (RAR) which, as activated by ATRA, selectively regulate gene expression. There are three main RAR isoforms, RARα, RARß, and RARγ. They each have a distinct role, for example, RARα and RARγ regulate myeloid progenitor cell differentiation and hematopoietic stem cell maintenance, respectively. Hence, targeting an isoform is crucial to developing retinoid-based therapeutics. In principle, this is exemplified when ATRA is used to treat acute promyelocytic leukemia (PML) and target RARα within PML-RARα oncogenic fusion protein. ATRA with arsenic trioxide has provided a cure for the once highly fatal leukemia. Recent in vitro and in vivo studies of RARγ have revealed the potential use of agonists and antagonists to treat diseases as diverse as cancer, heterotopic ossification, psoriasis, and acne. During the final drug development there may be a need to design newer compounds with added modifications to improve solubility, pharmacokinetics, or potency. At the same time, it is important to retain isotype specificity and activity. Examination of the molecular interactions between RARγ agonists and the ligand binding domain of RARγ has revealed aspects to ligand binding that are crucial to RARγ selectivity and compound activity and key to designing newer compounds.

Differential expression of retinoic acid alpha and beta receptors in neuronal progenitors generated from human embryonic stem cells in response to TTNPB (a retinoic acid mimetic).

Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the morphogenesis and differentiation of various tissues, especially in the central nervous system. RA is the most commonly used morphogen for the differentiation of human embryonic stem cells (hESCs) into neuronal progenitor cells (NPCs), an abundant source of healthy neuronal tissues for regenerative therapy. During the differentiation process, the activity of RA is governed by the involvement of RA receptor subtypes (RAR α, ß, and γ) and their isoforms in the nucleus. However, little is known about the involvement of specific RAR subtypes during neuronal differentiation in humans. It is essential to elucidate the dynamic function of different RAR subtypes and their influence on the phenotypic outcome. Here in this study, we used TTNPB, an analog and stabilized form of retinoic acid that potently and selectively activates retinoic acid receptors. Here we determined the optimum concentration of TTNPBfor the efficient generation of early NPCs from hESCs. Using the optimized concentration of -TTNPB, we found that RARα is the functionally dominant subtype and controls the RA-mediated neurogenesis of hESCs. Importantly, we also found that the RARγ subtype also played a role in neuronal differentiation. In contrast, the RARß subtype negatively correlates with neuronal differentiation. Therefore, pharmacological inhibition of RARß in the TTNPB-mediated differentiation process could be used as a strategy to generate a large number of NPCs in vitro. In summary, our results show that RARα and RARγ play a vital role in the TTNPB-mediated neuronal differentiation of hESCs, -whereas RARß acts as a negative regulator.

RARγ is required for mesodermal gene expression prior to gastrulation in Xenopus.

The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here, we show in Xenopus that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets upregulated by RA receptor signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the genes encoding the RA-synthesizing enzyme Aldh1a2 and the RA-degrading enzyme Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of Myod protein in the presomitic mesoderm, but ectopic, persistent expression of Myod protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation.

Ethanol promotes differentiation of embryonic stem cells through retinoic acid receptor-γ.

Ethanol (EtOH) is a teratogen, but its teratogenic mechanisms are not fully understood. The alcohol form of vitamin A (retinol/ROL) can be oxidized to all-trans-retinoic acid (RA), which plays a critical role in stem cell differentiation and development. Using an embryonic stem cell (ESC) model to analyze EtOH's effects on differentiation, we show here that EtOH and acetaldehyde, but not acetate, increase differentiation-associated mRNA levels, and that EtOH decreases pluripotency-related mRNAs. Using reporter assays, ChIP assays, and retinoic acid receptor-γ (RARγ) knockout ESC lines generated by CRISPR/Cas9 and homologous recombination, we demonstrate that EtOH signals via RARγ binding to RA response elements (RAREs) in differentiation-associated gene promoters or enhancers. We also report that EtOH-mediated increases in homeobox A1 (Hoxa1) and cytochrome P450 family 26 subfamily A member 1 (Cyp26a1) transcripts, direct RA target genes, require the expression of the RA-synthesizing enzyme, aldehyde dehydrogenase 1 family member A2 (Aldh1a2), suggesting that EtOH-mediated induction of Hoxa1 and Cyp26a1 requires ROL from the serum. As shown with CRISPR/Cas9 knockout lines, the retinol dehydrogenase gene Rdh10 and a functional RARE in the ROL transporter stimulated by retinoic acid 6 (Stra6) gene are required for EtOH induction of Hoxa1 and Cyp26a1 We conclude that EtOH stimulates stem cell differentiation by increasing the influx and metabolism of ROL for downstream RARγ-dependent transcription. In stem cells, EtOH may shift cell fate decisions to alter developmental outcomes by increasing endogenous ROL/RA signaling via increased Stra6 expression and ROL oxidation.

3-Cl-AHPC inhibits pro-HGF maturation by inducing matriptase/HAI-1 complex formation.

Matriptase is an epithelia-specific membrane-anchored serine protease, and its dysregulation is highly related to the progression of a variety of cancers. Hepatocyte growth factor activator inhibitor-1 (HAI-1) inhibits matriptase activity through forming complex with activated matriptase. The balance of matriptase activation and matriptase/HAI-1 complex formation determines the intensity and duration of matriptase activity. 3-Cl-AHPC, 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid, is an adamantly substituted retinoid-related molecule and a ligand of retinoic acid receptor γ (RARγ). 3-Cl-AHPC is of strong anti-cancer effect but with elusive mechanisms. In our current study, we show that 3-Cl-AHPC time- and dose- dependently induces matriptase/HAI-1 complex formation, leading to the suppression of activated matriptase in cancer cells and tissues. Furthermore, 3-Cl-AHPC promotes matriptase shedding but without increasing the activity of shed matriptase. Moreover, 3-Cl-AHPC inhibits matriptase-mediated cleavage of pro-HGF through matriptase/HAI-1 complex induction, resulting in the suppression of pro-HGF-stimulated signalling and cell scattering. Although 3-Cl-AHPC binds to RARγ, its induction of matriptase/HAI-1 complex is not RARγ dependent. Together, our data demonstrates that 3-Cl-AHPC down-regulates matriptase activity through induction of matriptase/HAI-1 complex formation in a RARγ-independent manner, providing a mechanism of 3-Cl-AHPC anti-cancer activity and a new strategy to inhibit abnormal matriptase activity via matriptase/HAI-1 complex induction using small molecules.

Different Effects of Knockouts in ALDH2 and ACSS2 on Embryonic Stem Cell Differentiation.

BACKGROUND: Ethanol (EtOH) is a teratogen that causes severe birth defects, but the mechanisms by which EtOH affects stem cell differentiation are unclear. Our goal here is to examine the effects of EtOH and its metabolites, acetaldehyde (AcH) and acetate, on embryonic stem cell (ESC) differentiation. METHODS: We designed ESC lines in which aldehyde dehydrogenase (ALDH2, NCBI#11669) and acyl-CoA synthetase short-chain family member 2 (ACSS2, NCBI#60525) were knocked out by CRISPR-Cas9 technology. We selected these genes because of their key roles in EtOH oxidation in order to dissect the effects of EtOH metabolism on differentiation. RESULTS: By using kinetic assays, we confirmed that AcH is primarily oxidized by ALDH2 rather than ALDH1A2. We found increases in mRNAs of differentiation-associated genes (Hoxa1, Cyp26a1, and RARß2) upon EtOH treatment of WT and Acss2-/- ESCs, but not Aldh2-/- ESCs. The absence of ALDH2 reduced mRNAs of some pluripotency factors (Nanog, Sox2, and Klf4). Treatment of WT ESCs with AcH or 4-hydroxynonenal (4-HNE), another substrate of ALDH2, increased differentiation-associated transcripts compared to levels in untreated cells. mRNAs of genes involved in retinoic acid (RA) synthesis (Stra6 and Rdh10) were also increased by EtOH, AcH, and 4-HNE treatment. Retinoic acid receptor-γ (RARγ) is required for both EtOH- and AcH-mediated increases in Hoxa1 and Stra6, demonstrating the critical role of RA:RARγ signaling in AcH-induced ESC differentiation. CONCLUSIONS: ACSS2 knockouts showed no changes in differentiation phenotype, while pluripotency-related transcripts were decreased in ALDH2 knockout ESCs. We demonstrate that AcH increases differentiation-associated mRNAs in ESCs via RARγ.

Structure-based design of Trifarotene (CD5789), a potent and selective RARγ agonist for the treatment of acne.

Retinoids have a dominant role in topical acne therapy and to date, only RARß and RARγ dual agonists have reached the market. Given the tissue distribution of RAR isoforms, it was hypothesized that developing RARγ -selective agonists could yield a new generation of topical acne treatments that would increase safety margins while maintaining the robust efficacy of previous drugs. Structural knowledge derived from the X-ray structure of known γ-selective CD437, suggested the design of a novel triaryl series of agonists which was optimized and ultimately led to the discovery of Trifarotene/CD5789.

Identification of potent and selective retinoic acid receptor gamma (RARγ) antagonists for the treatment of osteoarthritis pain using structure based drug design.

A series of triaryl pyrazoles were identified as potent pan antagonists for the retinoic acid receptors (RARs) α, ß and γ. X-ray crystallography and structure-based drug design were used to improve selectivity for RARγ by targeting residue differences in the ligand binding pockets of these receptors. This resulted in the discovery of novel antagonists which maintained RARγ potency but were greater than 500-fold selective versus RARα and RARß. The potent and selective RARγ antagonist LY2955303 demonstrated good pharmacokinetic properties and was efficacious in the MIA model of osteoarthritis-like joint pain. This compound demonstrated an improved margin to RARα-mediated adverse effects.

RARγ promotes the invasion and metastasis of thyroid carcinoma by activating the JAK1-STAT3-CD24/MMPs axis.

The nuclear receptor superfamily RAR is generally considered to play a crucial role in the development of tumors by regulating the transcription of target genes. Nevertheless, whether RARγ performs tumor-promoting or tumor-suppressing functions and its specific mechanism in thyroid carcinoma (TC) remain unknown. Here, our study demonstrated that RARγ was abnormally overexpressed in TC tissues compared with normal thyroid tissues. Moreover, RARγ expression was remarkably correlated with cell phenotypes such as cell proliferation, migration and invasion. Mechanistically, RARγ knockdown effectively decreased the phosphorylation levels of JAK1 and STAT3, leading to decreased expression of the membrane protein CD24. In a coculture system, TC cells with high levels of CD24 in the membrane were more likely to escape phagocytosis by macrophages via the combination of CD24 with the inhibitory receptor Siglec-10 in the membrane of macrophages. In contrast, the ability of macrophages to engulf TC cells was notably elevated through exogenous addition of CD24 antibody. Collectively, our study revealed a previously undiscovered molecular mechanism of RARγ in promoting the development of TC, shedding light on RARγ as a promising therapeutic target for TC.

Structural requirement of RARγ agonism through computational aspects.

CONTEXT: RARγ is a therapeutic target for many skin diseases and has potential in cancer treatment. In the current study, we put forward a comprehensive structure-activity relationship study of third and fourth generations of RARγ agonists, addressing multiple crystal structures of RARγ complexes and approved drugs. Adapalene and Trifarotene, through hybrid strategies including protein contacts Atlas analysis, molecular docking, dynamics simulations, MM-GBSA, ASM, and pharmacophore modeling. Our result revealed crucial amino acids Arg267, Ser278, Phe288, Phe230, Met272, Leu271, and Leu268 within the RARγ pocket, as well as pharmacophore features such as two hydrophobic groups, two aromatic rings, and negative ionic features, which are essential for the binding of RARγ agonists. Based on this study, the binding mechanism of RARγ agonists was elucidated, which will be helpful for the rational design of new RARγ agonists for skin diseases and cancer treatment. METHODS: In this study, Schrödinger suite 2021-2 with OPLS_4 force field, Discovery Studio program 3.0, LigandScout 4.3, and PyMOL are utilized in the investigation.

A synthetic retinoic acid receptor γ antagonist (7C)-loaded nanoparticle enhances bone morphogenetic protein-induced bone regeneration in a rat spinal fusion model.

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) have potent osteoinductivity and have been applied clinically for challenging musculoskeletal conditions. However, the supraphysiological doses of BMPs used in clinical settings cause various side effects that prevent widespread use, and therefore the BMP dosage needs to be reduced. PURPOSE: To address this problem, we synthesized 7C, a retinoic acid receptor γ antagonist-loaded nanoparticle (NP), and investigated its potential application in BMP-based bone regeneration therapy using a rat spinal fusion model. STUDY DESIGN: An experimental animal study. METHODS: Fifty-three male 8-week-old Sprague-Dawley rats underwent posterolateral spinal fusion and were divided into the following five treatment groups: (1) no recombinant human (rh)BMP-2 and blank-NP (Control), (2) no rhBMP-2 and 1 µg 7C-NP (7C group), (3) low-dose rhBMP-2 (0.5 µg) and 1 µg blank-NP (L-BMP group), (4) low-dose rhBMP-2 (0.5 µg) and 1 µg 7C-NP (L-BMP + 7C group), and (5) high-dose rhBMP-2 (5.0 µg) and 1 µg blank-NP (H-BMP group). Micro-computed tomography and histologic analysis were performed 2 and 6 weeks after the surgery. RESULTS: The spinal fusion rates of the Control and 7C groups were both 0%, and those of the L-BMP, L-BMP + 7C, and H-BMP groups were 55.6%, 94.4%, and 100%, respectively. The L-BMP + 7C group markedly promoted cartilaginous tissue formation during BMP-induced endochondral bone formation that resulted in a significantly better spinal fusion rate and bone formation than in the L-BMP group. Although spinal fusion was slower in the L-BMP + 7C group, the L-BMP + 7C group formed a spinal fusion mass with better bone quality than the spinal fusion mass in the H-BMP group. CONCLUSIONS: The combined use of 7C-NP with rhBMP-2 in a rat posterolateral lumbar fusion model increased spinal fusion rate and new bone volume without deteriorating the quality of newly formed bone. CLINICAL SIGNIFICANCE: 7C-NP potentiates BMP-2-induced bone regeneration and has the potential for efficient bone regeneration with low-dose BMP-2, which can reduce the dose-dependent side effects of BMP-2 in clinical settings.

Selective Retinoic Acid Receptor γ Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation.

Bone morphogenetic proteins (BMPs) have been clinically applied for induction of bone formation in musculoskeletal disorders such as critical-sized bone defects, nonunions, and spinal fusion surgeries. However, the use of supraphysiological doses of BMP caused adverse events, which were sometimes life-threatening. Therefore, safer treatment strategies for bone regeneration have been sought for decades. Systemic administration of a potent selective antagonist of retinoic acid nuclear receptor gamma (RARγ) (7C) stimulated BMP-induced ectopic bone formation. In this study, we developed 7C-loaded poly lactic nanoparticles (7C-NPs) and examined whether local application of 7C enhances BMP-induced bone regeneration. The collagen sponge discs that absorbed recombinant human (rh) BMP-2 were implanted into the dorsal fascia of young adult mice to induce ectopic bone. The combination of rhBMP-2 and 7C-NP markedly increased the total bone volume and thickness of the bone shell of the ectopic bone in a dose-dependent manner compared to those with rhBMP-2 only. 7C stimulated sulfated proteoglycan production, expression of chondrogenic marker genes, and Sox9 reporter activity in both chondrogenic cells and MSCs. The findings suggest that selective RARγ antagonist 7C or the related compounds potentiate the bone inductive ability of rhBMP-2, as well as support any future research to improve the BMP-2 based bone regeneration procedures in a safe and efficient manner.

Reduced Retinoic Acid Receptor Beta (Rarß) Affects Pancreatic ß-Cell Physiology.

Various studies have suggested a link between vitamin A (VA), all-trans-retinol, and type 2 diabetes (T2D). However, the functional role/expression of vitamin A receptors (Rarα, ß, and γ) in pancreatic ß-cells is not clear yet. Accordingly, we performed a series of bioinformatics, molecular and functional experiments in human islet and INS-1 cells to evaluate the role of Rarß on insulin secretion and pancreatic ß-cell function. Microarray and RNA-sequencing (RAN-seq) expression analysis showed that RARα, ß, and γ are expressed in human pancreatic islets. RNA-seq expression of RARß in diabetic/hyperglycemic human islets (HbA1c ≥ 6.3%) revealed a significant reduction (p = 0.004) compared to nondiabetic/normoglycemic cells (HbA1c < 6%). The expression of RARß with INS and PDX1 showed inverse association, while positive correlations were observed with INSR and HbA1c levels. Exploration of the T2D knowledge portal (T2DKP) revealed that several genetic variants in RARß are associated with BMI. The most associated variant is rs6804842 (p = 1.2 × 10−25). Silencing of Rarß in INS-1 cells impaired insulin secretion without affecting cell viability or apoptosis. Interestingly, reactive oxygen species (ROS) production levels were elevated and glucose uptake was reduced in Rarß-silenced cells. mRNA expression of Ins1, Pdx1, NeuroD1, Mafa, Snap25, Vamp2, and Gck were significantly (p < 0.05) downregulated in Rarß-silenced cells. For protein levels, Pro/Insulin, PDX1, GLUT2, GCK, pAKT/AKT, and INSR expression were downregulated considerably (p < 0.05). The expression of NEUROD and VAMP2 were not affected. In conclusion, our results indicate that Rarß is an important molecule for ß-cell function. Hence, our data further support the potential role of VA receptors in the development of T2D.

Transition from TNF-Induced Inflammation to Death Signaling.

Tumor necrosis factor (TNF) plays a key role in inflammatory responses and in various cellular events such as apoptosis and necroptosis. The interaction of TNF with its receptor, TNFR1, drives the initiation of complex molecular pathways leading to inflammation and cell death. RARγ is released from the nucleus to orchestrate the formation of the cytosolic death complexes, and it is cytosolic RARγ that plays a pivotal role in switching TNF-induced inflammatory responses to RIPK1-initiated cell death. Thus, RARγ provides a checkpoint for the transition from inflammatory signaling to death machinery of RIPK1-initiated cell death in response to TNF. Here, we use techniques to identify RARγ as a downstream mediator of TNFR1 signaling complex. We use confocal imaging to show the localization of RARγ upon activation of cell death. Immunoprecipitation of RARγ identified the interacting proteins.

Retinoic acid receptor γ is a therapeutically targetable driver of growth and survival in prostate cancer.

BACKGROUND: Prostate cancer (PC) tissue contains all-trans retinoic acid (ATRA) at a very low level (10-9 M), at least an order of magnitude lower than in adjacent normal healthy prostate cells or benign prostate hyperplasia. When this is coupled with deregulated expression of the intracellular lipid-binding proteins FABP5 and CRABP2 that is frequently found in PC, this is likely to result in the preferential delivery of ATRA to oncogenic PPARß/δ rather than retinoic acid receptors (RARs). There are three isotypes of RARs (RARα, RARß, and RARγ) and recent studies have revealed discrete physiological roles. For example, RARα and RARγ promote differentiation and self-renewal, respectively, which are critical for proper hematopoiesis. AIMS: We have previously shown that ATRA stimulates transactivation of RARγ at sub-nanomolar concentrations (EC50 0.24 nM), whereas an 80-fold higher concentration was required for RARα-mediated transactivation (EC50 19.3 nM). Additionally, we have shown that RAR pan-antagonists inhibit the growth of PC cells (at 16-34 nM). These findings, together with the low level of ATRA in PC, led us to hypothesize that RARγ plays a role in PC pathogenesis and that RARγ-selective antagonism may be an effective treatment. METHODS AND RESULTS: We found that concentrations of 10-9 M and below of ATRA promoted survival/proliferation and opposed adipogenic differentiation of human PC cell lines by a mechanism that involves RARγ. We also found that a RARγ-selective antagonist (AGN205728) potently induced mitochondria-dependent, but caspase-independent, cell death in PC cell lines. Furthermore, AGN205728 demonstrated synergism in killing PC cells in combination with cytotoxic chemotherapeutic agents. CONCLUSION: We suggest that the use of RARγ-selective antagonists may be effective in PC (and potentially other cancers), either as a single agent or in combination with cytotoxic chemotherapy.

A novel tsRNA-16902 regulating the adipogenic differentiation of human bone marrow mesenchymal stem cells.

BACKGROUND: Transfer RNA-derived small RNAs (tsRNAs) are a recently discovered form of non-coding RNA capable of regulating myriad physiological processes. The role of tsRNAs in hMSC adipogenic differentiation, however, remains incompletely understood. The purpose of this study was to identify the novel tsRNA-16902 as a regulator of hMSC adipogenic differentiation. METHODS: In this study, we conducted transcriptomic sequencing of hMSCs after inducing their adipogenic differentiation, and we were thereby able to clarify the molecular mechanism underlying the role of tsRNA-16902 in this context via a series of molecular biology methods. RESULTS: When we knocked down tsRNA-16902 expression, this impaired hMSC adipogenic differentiation and associated marker gene expression. Bioinformatics analyses further revealed tsRNA-16902 to target retinoic acid receptor γ (RARγ). Luciferase reporter assays also confirmed the ability of tsRNA-16902 to bind to the RARγ 3'-untranslated region. Consistent with this, RARγ overexpression led to impaired hMSC adipogenesis. Further analyses revealed that Smad2/3 phosphorylation was increased in cells that either overexpressed RARγ or in which tsRNA-16902 had been knocked down. We also assessed the adipogenic differentiation of hMSCs in which tsRNA-16902 was knocked down and at the same time a Smad2/3 inhibitor was added to disrupt Smad2/3 phosphorylation. The adipogenic differentiation of hMSCs in which tsRNA-16902 was knocked down was further enhanced upon the addition of a Smad2/3 signaling inhibitor relative to tsRNA-16902 knockdown alone. CONCLUSIONS: Through a comprehensive profiling analysis of tsRNAs that were differentially expressed in the context of hMSC adipogenic differentiation, we were able to identify tsRNA-16902 as a previously uncharacterized regulator of adipogenesis. tsRNA-16902 is able to regulate hMSC adipogenic differentiation by targeting RARγ via the Smad2/3 signaling pathway. Together, our results may thus highlight novel strategies of value for treating obesity.

Response to comment on 'Palovarotene reduces heterotopic ossification in juvenile FOP mice but exhibits pronounced skeletal toxicity'.

We respond to concerns expressed by Pacifici and Shore (2019) about a recent paper (Lees-Shepard and Goldhamer, 2018a) in which we reported that the drug palovarotene can have severe side effects in a mouse model of fibrodysplasia ossificans progressiva.

Therapeutic potential of Bcl-xL/Mcl-1 synthetic inhibitor JY-1-106 and retinoids for human triple-negative breast cancer treatment.

Overexpression of anti-apoptotic proteins belonging to the B cell lymphoma (Bcl)-2 family is observed in numerous cancer types and has been postulated to promote cancer cell survival and chemotherapy resistance. Bcl-extra large (xL)/myeloid cell leukemia sequence (Mcl)-1 was demonstrated to be expressed at relatively high levels in clinically aggressive basal-like cancers and inhibiting Bcl-xL overexpression could potentially provoke cell death. A molecule able to target Bcl-xL/Mcl-1, JY-1-106, is herein under investigation. It is also known that vitamin A-derived compounds exhibit antitumor activity in a variety of in vitro experimental models, promoting their effects via nuclear receptor isoforms including retinoic acid receptors (RARs). Pre-clinical observation highlighted that triple negative (estrogen receptor/progesterone receptor/human epidermal growth factor receptor)-breast cancer cells displayed resistance to retinoids due to the RARγ high expression profile. The present study used the triple-negative human breast cancer cell line, MDA-MB-231, to analyze the effects of the Bcl-xL/Mcl-1 synthetic inhibitor, JY-1-106, alone or in combination with retinoids on cell viability. The results revealed a synergistic effect in reducing cell viability primarily by using JY-1-106 with the selective RARγ antagonist SR11253, which induces massive autophagy and necrosis. Furthermore, the results highlighted that JY-1-106 alone is able to positively influence the gene expression profile of p53 and RARα, providing a therapeutic advantage in human triple-negative breast cancer treatment.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP): A stage-specific biomarker of heterotopic endochondral ossification (HEO) in fibrodysplasia ossificans progressiva (FOP).

BACKGROUND: Genesis of a cartilaginous scaffold is an obligate precursor to bone formation in heterotopic endochondral ossification (HEO). We tested the hypothesis that cartilage-derived retinoic acid-sensitive protein (CD-RAP) can serve as a plasma biomarker for the pre-osseous cartilaginous stage of HEO. Palovarotene, a retinoic acid receptor-gamma (RARγ) agonist, has been proposed as a possible treatment for fibrodysplasia ossificans progressiva (FOP) and is a potent inhibitor of HEO in mouse models. Current drug development for FOP mandates the identification of stage-specific biomarkers to facilitate the evaluation of clinical trial endpoints. RESULTS: Here we show in an injury-induced, constitutively-active transgenic mouse model of FOP that CD-RAP levels peaked between day-7 and day-10 during the zenith of histologically-identified chondrogenesis, preceded radiographically apparent HEO, and were diminished by palovarotene. Cross-sectional analysis of CD-RAP levels in plasma samples from FOP patients demonstrated a statistically non-significant trend toward higher levels in the recent flare-up period (three weeks to three months within onset of symptoms). However, in a longitudinal subgroup analysis of patients followed for at least six months after resolution of flare-up symptoms, there was a statistically significant decrease of CD-RAP when compared to levels in the same patients at the time of active or recent exacerbations. CONCLUSIONS: These data support the further exploration of CD-RAP as a stage-specific biomarker of HEO in FOP.