Homeobox Protein Research Articles

Genome-wide association study identified genes associated with ammonia nitrogen tolerance in Litopenaeus vannamei.

Ammonia nitrogen tolerance is an economically important trait of the farmed penaeid shrimp Litopenaeus vannamei. To identify the genes associated with ammonia nitrogen tolerance, we performed an extreme phenotype genome-wide association study method (XP-GWAS) on a population of 200 individuals. The single nucleotide polymorphism (SNP) genotyping array method was used to construct the libraries and 36,048 SNPs were genotyped. Using the MLM, FarmCPU and Blink models, six different SNPs, located on SEQ3, SEQ4, SEQ5, SEQ7 and SEQ8, were determined to be significantly associated with ammonia nitrogen tolerance. By integrating the results of the GWAS and the biological functions of the genes, seven candidate genes (PDI, OZF, UPF2, VPS16, TMEM19, MYCBP2, and HOX7) were found to be associated with ammonia nitrogen tolerance in L. vannamei. These genes are involved in cell transcription, cell division, metabolism, and immunity, providing the basis for further study of the genetic mechanisms of ammonia nitrogen tolerance in L. vannamei. Further candidate gene association analysis in the offspring population revealed that the SNPs in the genes zinc finger protein OZF-like (OZF) and homeobox protein Hox-B7-like (HOX7) were significantly associated with ammonia nitrogen tolerance trait of L. vannamei. Our results provide fundamental genetic information that will be useful for further investigation of the molecular mechanisms of ammonia nitrogen tolerance. These associated SNPs may also be promising candidates for improving ammonia nitrogen tolerance in L. vannamei.

Genome-Wide Analysis of the WOX Transcription Factor Genes in Dendrobium catenatum Lindl.

The WUSCHEL-related homeobox (WOX) proteins are a class of transcription factors exclusive to plants. They can promote cell division or inhibit stem cell differentiation to regulate plant growth and development. However, the WOX transcription factor genes in the monocotyledon Dendrobium catenatum Lindl. remain relatively uncharacterized. Specifically, the effects of phytohormones on their expression levels are unclear. In this study, we identified and analyzed 10 candidate DcaWOX transcription factor genes in D. catenatum. The DcaWOX family was divided into the modern/WUS, intermediate, and ancient clades. The subcellular localization analysis detected DcaWOX-GFP fusion proteins in the tobacco epidermal leaf cell nucleus. In DcaWOX, members of the WUS clade with the WUS-box motif can significantly activate the expression of TPL in vivo, while members of the intermediate and ancient clades cannot. The expression of the DcaWOX genes varied among the examined tissues. Moreover, the DcaWOX expression patterns were differentially affected by the phytohormone treatments, with differences detected even between homologs of the same gene. Furthermore, the gene expression patterns were consistent with the predicted cis-acting elements in the promoters. The above results suggest that DcaWOX may have an important role in its growth and development and resistance to stress. The results of this comprehensive investigation of the DcaWOX gene family provide the basis for future studies on the roles of WOX genes in D. catenatum.

IRX3 plays an important role in the pathogenesis of metabolic-associated fatty liver disease by regulating hepatic lipid metabolism.

Metabolic-associated fatty liver disease (MAFLD) affects approximately a quarter of the global population. Identification of the key genes and pathways involved in hepatic lipid metabolism is of the utmost importance for the diagnosis, treatment, and prevention of MAFLD. In this study, differentially expressed genes were identified through whole-genome transcriptional analysis of liver tissue from MAFLD patients and healthy controls, and a series of lipid metabolism-related molecules and pathways were obtained through pathway analysis. Subsequently, we focused on Iroquois homeobox protein 3 (IRX3), one of 13 transcription factors that were screened from the 331 differentially expressed genes. The transcription factor IRX3 was significantly decreased in the liver tissue of patients with MAFLD when compared with healthy controls. Pearson’s correlation analysis showed that the expression levels of IRX3 in liver tissue were negatively correlated with serum total cholesterol, triglycerides, low-density lipoprotein cholesterol, and uric acid levels. The overexpression and interference of IRX3 induced the increased and decreased lipid droplet accumulation in vitro, respectively. Moreover, interference of IRX3 expression increased mitochondrial fragmentation and reduced the activity of the mitochondrial respiratory chain complex IV. In summary, the study demonstrated that IRX3 regulated hepatic lipid metabolism of MAFLD, and also revealed the effect of IRX3 on mitochondria might be an important mechanism by which IRX3 regulated hepatic lipid metabolism of MAFLD.

Circ_0010235 Regulates HOXA10 Expression to Promote Malignant Phenotypes and Radioresistance in Non-small Cell Lung Cancer Cells Via Decoying miR-588.

Background:Circular RNAs (circRNAs) are key modulators in carcinogenesis and radioresistance in multiple kinds of human cancers.Aims:To explore the role of circ_0010235 in non-small cell lung cancer (NSCLC).Study Design:Cell culture study and animal study.Methods:The detection of circ_0010235, microRNA-588 (miR-588), and homeobox protein A10 (HOXA10) was implemented via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). CCK-8, EdU, flow cytometry, transwell, and wound healing assays. These strategies were applied to evaluate cell functions. The western blot technique was employed for protein examination. The colony formation assay was used to determine cell survival after radiation treatment. In vivo research was performed by tumor xenograft assay. The binding analysis was also carried out through dual-luciferase reporter and RNA immunoprecipitation studies.Results:Circ_0010235 had an enhanced expression in NSCLC. Circ_0010235 deficiency inhibited cell proliferation, invasiveness, and migratory ability but promoted apoptosis and radiosensitivity. Downregulation of circ_0010235 decelerated tumor growth and promoted radiation sensitivity in vivo. Circ_0010235 was controlled biologically in NSCLC cells by combining with miR-588 and targeting miR-588. HOXA10 acted as a target of miR-588. MiR-588 upregulation inhibited NSCLC cell malignant phenotypes and elevated radiosensitivity via downregulating HOXA10. Circ_0010235 could regulate the level of HOXA10 by sponging miR-588.Conclusion:Circ_0010235 contributed to the malignant progression of NSCLC, but suppressed the radiation sensitivity via targeting miR-588 to induce HOXA10 upregulation.

Homeobox 27, a Homeodomain Transcription Factor, Confers Tolerances to CMV by Associating with Cucumber Mosaic Virus 2b Protein.

Transcription factors (TFs) play an important role in plant development; however, their role during viral infection largely remains unknown. The present study was designed to uncover the role transcription factors play in Cucumber mosaic virus (CMV) infection. During the screening of an Arabidopsis thaliana (Col-0) transcription factor library, using the CMV 2b protein as bait in the yeast two-hybrid system, the 2b protein interacted with Homeobox protein 27 (HB27). HB27 belongs to the zinc finger homeodomain family and is known to have a regulatory role in flower development, and responses to biotic and abiotic stress. The interaction between CMV 2b and HB27 proteins was further validated using in planta (bimolecular fluorescence complementation assay) and in vitro far-Western blotting (FWB) methods. In the bimolecular fluorescence complementation assay, these proteins reconstituted YFP fluorescence in the nucleus and the cytoplasmic region as small fluorescent dots. In FWB, positive interaction was detected using bait anti-MYC antibody on the target HB27-HA protein. During CMV infection, upregulation (~3-fold) of the HB27 transcript was observed at 14 days post-infection (dpi) in A. thaliana plants, and expression declined to the same as healthy plants at 21 dpi. To understand the role of the HB27 protein during CMV infection, virus accumulation was determined in HB27-overexpressing (HB27 OE) and knockout mutants. In HB27-overexpressing lines, infected plants developed mild symptoms, accumulating a lower virus titer at 21 dpi compared to wild-type plants. Additionally, knockout HB27 mutants had more severe symptoms and a higher viral accumulation than wild-type plants. These results indicate that HB27 plays an important role in the regulation of plant defense against plant virus infection.

NFIA determines the cis-effect of genetic variation on Ucp1 expression in murine thermogenic adipocytes

SummaryThermogenic brown and beige adipocytes counteract obesity by enhancing energy dissipation via uncoupling protein-1 (Ucp1). However, the effect of genetic variation on these cells, a major source of disease susceptibility, has been less well studied. Here we examined beige adipocytes from obesity-prone C57BL/6J (B6) and obesity-resistant 129X1/SvJ (129) mouse strains and identified a cis-regulatory variant rs47238345 that is responsible for differential Ucp1 expression. The alternative T allele of rs47238345 at the Ucp1 -12kb enhancer in 129 facilitates the allele-specific binding of nuclear factor I-A (NFIA) to mediate allele-specific enhancer-promoter interaction and Ucp1 transcription. Furthermore, CRISPR-Cas9/Cpf1-mediated single nucleotide polymorphism (SNP) editing of rs47238345 resulted in increased Ucp1 expression. We also identified Lim homeobox protein 8 (Lhx8), whose expression is higher in 129 than in B6, as a trans-acting regulator of Ucp1 in mice and humans. These results demonstrate the cis- and trans-acting effects of genetic variation on Ucp1 expression that underlie phenotypic diversity.

Molecular Characterization of Tomato (Solanum lycopersicum L.) Accessions under Drought Stress

Exploring the genetic diversity among plant accessions is important for conserving and managing plant genetic resources. In the current study, a collection of forty-six tomato accessions from Jordan were evaluated based on their performance and their morpho-physiological, in addition to molecularly characterizing to detect genetic diversity. Tomato accessions seedlings were exposed to drought stress with 70% field capacity and 40% field capacity under field conditions in Jordan. Drought stress had significantly negatively influenced the dry root weight, fresh root weight, root growth rate, fresh shoot weight, dry shoot weight, and shoot growth rate. Moreover, proline content showed a highly significant increase of 304.2% in response to drought stress. The analysis of twenty morphological characters revealed a wide range of variations among tomato accessions. Accessions were screened with fourteen SSR primers; six primers were informative to explain the genetic diversity. Based on resolving power, primers LEct004 and LEat018 were most significant with all 46 accessions. Interestingly, polymorphic information content (PIC) values ranged from 0.00 (Asr2 marker) to 0.499 (LEct004), which confirms that the SSR markers are highly informative. Our findings provide new insights into using informative molecular markers to elucidate such wide genetic variation discovered in our collections from Afraa and Abeel (the southern part of Jordan). Interestingly, the SSR markers were associated with genes, e.g., LEat018 with ACTIN_RELATED PROTEIN gene, the LEct004 with the HOMEOBOX PROTEIN TRANSCRIPTION FACTORS gene, and Asr2 with ABA/WDS. Moreover, the AUXIN RESPONSE FACTOR8 gene was associated with the LEta014 SSR marker and the LEta020 with the THIOREDOXIN FAMILY TRP26 gene. Therefore, the genetic diversity analysis and functional annotations of the genes associated with SSR information obtained in this study provide valuable information about the most suitable genotype that can be implemented in plant breeding programs and future molecular analysis. Furthermore, evaluating the performance of the collection under different water regimes is essential to produce new tomato varieties coping with drought stress conditions.

Genome-Wide Identification and Comparative Analysis of WOX Genes in Four Euphorbiaceae Species and Their Expression Patterns in Jatropha curcas.

The WUSCHEL-related homeobox (WOX) proteins are widely distributed in plants and play important regulatory roles in growth and development processes such as embryonic development and organ development. Here, series of bioinformatics methods were utilized to unravel the structural basis and genetic hierarchy of WOX genes, followed by regulation of the WOX genes in four Euphorbiaceae species. A genome-wide survey identified 59 WOX genes in Hevea brasiliensis (H. brasiliensis: 20 genes), Jatropha curcas (J. curcas: 10 genes), Manihot esculenta (M. esculenta: 18 genes), and Ricinus communis (R. communis: 11 genes). The phylogenetic analysis revealed that these WOX members could be clustered into three close proximal clades, such as namely ancient, intermediate and modern/WUS clades. In addition, gene structures and conserved motif analyses further validated that the WOX genes were conserved within each phylogenetic clade. These results suggested the relationships among WOX members in the four Euphorbiaceae species. We found that WOX genes in H. brasiliensis and M. esculenta exhibit close genetic relationship with J. curcas and R. communis. Additionally, the presence of various cis-acting regulatory elements in the promoter of J. curcas WOX genes (JcWOXs) reflected distinct functions. These speculations were further validated with the differential expression profiles of various JcWOXs in seeds, reflecting the importance of two JcWOX genes (JcWOX6 and JcWOX13) during plant growth and development. Our quantitative real-time PCR (qRT-PCR) analysis demonstrated that the JcWOX11 gene plays an indispensable role in regulating plant callus. Taken together, the present study reports the comprehensive characteristics and relationships of WOX genes in four Euphorbiaceae species, providing new insights into their characterization.

Integrated Analyses of DNA Methylation and Gene Expression of Rainbow Trout Muscle under Variable Ploidy and Muscle Atrophy Conditions.

Rainbow trout, Oncorhynchus mykiss, is an important cool, freshwater aquaculture species used as a model for biological research. However, its genome reference has not been annotated for epigenetic markers affecting various biological processes, including muscle growth/atrophy. Increased energetic demands during gonadogenesis/reproduction provoke muscle atrophy in rainbow trout. We described DNA methylation and its associated gene expression in atrophying muscle by comparing gravid, diploid females to sterile, triploid females. Methyl Mini-seq and RNA-Seq were simultaneously used to characterize genome-wide DNA methylation and its association with gene expression in rainbow trout muscle. Genome-wide enrichment in the number of CpGs, accompanied by depleted methylation levels, was noticed around the gene transcription start site (TSS). Hypermethylation of CpG sites within ±1 kb on both sides of TSS (promoter and gene body) was weakly/moderately associated with reduced gene expression. Conversely, hypermethylation of the CpG sites in downstream regions of the gene body +2 to +10 kb was weakly associated with increased gene expression. Unlike mammalian genomes, rainbow trout gene promotors are poor in CpG islands, at <1% compared to 60%. No signs of genome-wide, differentially methylated (DM) CpGs were observed due to the polyploidy effect; only 1206 CpGs (0.03%) were differentially methylated, and these were primarily associated with muscle atrophy. Twenty-eight genes exhibited differential gene expression consistent with methylation levels of 31 DM CpGs. These 31 DM CpGs represent potential epigenetic markers of muscle atrophy in rainbow trout. The DM CpG-harboring genes are involved in apoptosis, epigenetic regulation, autophagy, collagen metabolism, cell membrane functions, and Homeobox proteins. Our study also identified genes explaining higher water content and modulated glycolysis previously shown as characteristic biochemical signs of rainbow trout muscle atrophy associated with sexual maturation. This study characterized DNA methylation in the rainbow trout genome and its correlation with gene expression. This work also identified novel epigenetic markers associated with muscle atrophy in fish/lower vertebrates.

P1310: A ROBUST APPROACH FOR THE GENERATION OF FUNCTIONAL HEMATOPOIETIC PROGENITOR CELL LINES TO MODEL LEUKEMIC TRANSFORMATION

Background: Preclinical research aims at developing robust methods to maintain, expand and manipulate haematopoietic stem/progenitor cells (HSPC) ex vivo, as these cells are infrequent and their immortalization is hardly achieved. The transcription factor, LIM homeobox protein 2 (LHX2), has a critical role in maintaining self-renewal and has been reported to facilitate ex vivo expansion of immature HSPCs. Aims: We aimed to establish a robust and low-cost murine HSPC in vitro model system that allows maintenance and expansion of functional LSK (lin-, Sca-1+, c-Kit+) cells, and guarantees long-term multipotency. Methods: We generated HPCLSK cell lines from high-purity sorted murine LSK cells by Lhx2 overexpression. SCF/IL-6 dependent HPCLSK cells are kept on agarose-coated plates to prevent adherence-induced myeloid differentiation. HPCLSK cells can be genetically modified e.g. by retroviral transduction, which serves as a source to generate LSCs harboring hematopoietic stem cell oncogenes including BCR/ABLp210+, MLL-AF9 or FLT3-ITD/NRASG12D. Immunophenotyping, transcriptome analysis and functionality tests of HPCLSK and LSC lines were carried out. Results: HPCLSK cells preserve LSK markers despite continuous proliferation. HPCLSK cells efficiently home to the BM, blood, spleen, and thymus and are able to differentiate into myeloid and lymphoid lineages in vitro and in vivo. Numbers of HPCLSK–derived myeloid and lymphoid progenitors in the BM and differentiated blood cells are comparable to BM-injected mice. Transcriptome analysis of HPCLSK lines showed a ST-HSC/MPP1 signature, which corresponds to the earliest proliferating HSPCs. Injection of transformed HPCLSK-LSC (BCR/ABLp210, MLL-AF9, FLT3-ITD/NRASG12D) lines in immunocompromised mice induced myeloid leukemia. All diseased animals displayed elevated WBC counts, blast-like cells in the blood and suffered from splenomegaly. To test the applicability of the model system for drug sensitivity tests we employed Imatinib mesylate (IM). As in patients, HPCLSK-BCR/ABLp210 cells show partial IM resistance and leads to the enrichment of undifferentiated LSK cells. Summary/Conclusion: We created a robust method of culturing functional HSPCs, which are immortalized and can be expanded indefinitely. HPCLSK lines can be rapidly generated from transgenic mouse models and are a useful system for functional assays that require high cell numbers. By oncogenic transformation we created leukemia triggering LSCs. These findings support the relevance of HPCLSK cell system that represents a unique tool to compare LSCs to non-transformed HPCLSK cells in vitro and in vivo, which can be adapted for high-scale preclinical compound screening.

The Landscape of DNA Methylation Generates Insight Into Epigenetic Regulation of Differences Between Slow-Twitch and Fast-Twitch Muscles in Pseudocaranx dentex

Skeletal muscles of teleost are mainly composed of slow-twitch muscles (SM) and fast-twitch muscles (FM) differed in contractile properties, metabolic capacities, and regeneration rate. The transcriptional regulatory mechanisms that control different muscle types have been elucidated in teleost according to transcriptome between SM and FM. However, the differences between SM and FM were affected not only by genotype but also by complicated epigenetic effects, including DNA methylation, which usually regulates genes in transcription level. To determine the essential role of DNA methylation in the regulation of different muscle types, we analyzed whole-genome methylation profiles of pelagic migratory fish Pseudocaranx dentex with abundant and well-separated SM and integrated DNA methylation profiles with the previously obtained transcriptome data. A total of 4,217 differentially methylated genes (DMGs) were identified, of which 3,582 were located in the gene body and 635 in the promoter. These DMGs mainly participated in muscle metabolite and cell junction. Enriched cell junction pathway reflected different capillary distribution between SM and FM. Through comprehensive analysis of methylome and transcriptome, 84 differentially expressed genes (DEGs) showed significant methylation variation in promoters between SM and FM, indicating that their expression was regulated by DNA methylation. Hypomethylated and highly expressed oxygen storage protein Myoglobin (myg) in SM indicated demethylation of myg promoter could upregulate its expression, thus increasing O2 supplying and meeting oxygen demands of SM. Hypermethylated and lowly expressed tnn (Troponin) and rlc (myosin regulatory light chain) in SM may be associated with low mobility of myosin cross bridges, which lead to slower and less frequent muscle contraction in SM than in FM. In addition, hypomethylated and highly expressed lbx1 (Ladybird homeobox protein homolog 1) and epo (erythropoietin) may be related to increased satellite cell numbers, and Semaphorin/Plexin genes may be related to higher rate of neuromuscular connection reconstruction, which further promote high muscle regeneration efficiency in SM. Our study elucidated the potential DNA methylation mechanisms that regulate physiological characteristics differences between SM and FM, which could facilitate our understanding of skeletal muscle adaptation in pelagic migratory fishes and further enrich the theoretical basis for the study of physiological characteristics and adaptive evolution in teleost fishes.

Abstract 1423: NKX3.1 expression predicts response to metformin intervention in prostate cancer

Abstract The goal of precision prevention is to tailor interventions based on individualized patient cancer risk and likelihood of progression. This approach is particularly relevant for prostate cancer since it is characteristically indolent, and thus delaying disease progression can be highly advantageous for patients. Therefore, identifying molecular features predictive of cancer progression and response to specific interventions is vital to improve precision prevention in prostate cancer. In this regard, expression levels and localization of the tissue-specific homeobox protein, NKX3.1, helps distinguish men with high risk for developing more aggressive prostate cancer. Our recent study revealed a novel function for NKX3.1 in mitochondria, where it regulates aberrant mitochondrial oxidative stress to suppress prostate cancer initiation. Lower NKX3.1 expression levels at gene and protein levels are associated with poor outcome and higher risk for aggressive prostate cancer. We are now investigating the role of NKX3.1 as a prognostic marker and potential target for prostate cancer precision prevention interventions. Our new preliminary data indicate a role for metformin as a preventative intervention in contexts of reduced NKX3.1. Specifically, we have shown that metformin reduces oxidative stress and suppresses cancer initiation in the prostates of Nkx3.1 mutant mice. Moreover, studies in human prostate cancer cells as well as human prostate cancer organotypic assays revealed that metformin can indeed, restore mitochondrial function and inhibit tumorigenicity in the absence of NKX3.1. Although men on active surveillance for prostate cancer may benefit from metformin intervention, it has been unclear whether metformin is effective in delaying disease progression and, if so, whether specific patients are more likely to benefit. Our new data implicate NKX3.1 status as a predictor of response to metformin, suggesting that it can stratify high-risk prostate cancer patients most likely to benefit from such intervention during active surveillance. Citation Format: Alexandros Papachristodoulou, Aditya Dutta, Cory Abate-Shen. NKX3.1 expression predicts response to metformin intervention in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1423.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

Expression of the Homeobox Proteins Dlx-5 and TLX1 During the Development of Cat Testis

Recent studies have shown that homeobox proteins play an important role in the formation and development of tissues and organs in the embryonic period. In our study, the distribution of Dlx-5 and TLX proteins, which are members of the homeobox family, in the testis, epididymis and ductus deferens ducts of some cat breeds were investigated. For this purpose, in the study, 18 testes younger than six months (immature) and older than one year (mature) were examined under a light microscope using an immunohistochemical method (indirect streptavidin-biotin complex). While it was determined that Dlx-5 and TLX1 proteins were expressed at varying levels in cells in immature and mature cat testicles, epithelial cells of ductus epididymis and ductus deferens, and smooth muscle cells of ductus deferens, no differences were observed between cat breeds. Dlx-5 immunoreactivity was more intense in the testes, epididymis and deferens ducts of immature and mature compared to TLX1. These results suggested that both proteins play important roles in the development of male feline genital organs and in the secretion and differentiation of cells, and also further observation of Dlx-5 expression suggested that this protein may be more effective than TLX1 in testicular development and physiological processes.

The Relationship of iTILs (Intratumoral Infiltrating Lymphocytes) and sTILs (Stromal Infiltrating Lymphocytes) with SOX2 (Sex-Determining Region Y (SRY)-Box2) Expression in Nasopharyngeal Carcinoma

Background: Nasopharyngeal carcinoma (NPC) is a type of malignant head and neck tumor originating from the epithelial lining of the nasopharynx which is commonly found in Asian countries, including Indonesia. SOX2 is part of SOX. The SOX gene family first emerged with the discovery of the mammalian testicular determinant, Sry. SOX2 expression in NPC is promoted by transcription factors such as activation protein 2 (AP-2), homeobox protein 1 (Prox1) and Pax6. TILs can be used systematically to evaluate a patient's anti-cancer immunological function that can be a valuable prognostic factor. Research Objectives: To determine the relationship between the degree of iTILs (intratumoral infiltrating lymphocytes) and sTILs (stromal infiltrating lymphocytes) with the immunohistochemical expression of SOX2 (sex-determining region Y (SRY)-box 2) in the histopathology of nasopharyngeal carcinoma. Materials and Methods: 43 samples in the form of a histopathological slide from biopsy/operative tissue diagnosed with nasopharyngeal carcinoma were collected. Stromal and intratumoral TILs were evaluated by the researcher and two anatomic pathologists, then immunohistochemical staining of SOX2 was performed and the expression was assessed. Spearman correlation test was performed to analyse between variables. Results: Based on the Spearman?s correlation test, there was no significant relationship between the degree of iTILs and SOX2 expression (p= 0.892) and there was a significant relationship between the degree of sTILs and SOX2 expression (p= 0.003). Conclusion: This study shows no significant relationship between the degree of iTILs and SOX2 expression, but there was a significant relationship between the degree of sTILs and SOX2 expression.

Lerociclib diminishes stemness in pediatric sarcoma cell lines.

e22010 Background: The outcomes for children with osteosarcoma and synovial sarcoma remain poor and are even worse for metastatic or relapsed disease. Those who do survive frequently suffer from long-term toxicities from current standard-of-care therapies. It is evident novel therapeutics are warranted to improve the outcomes for patients with these difficult tumors. Stem cell-like cancer cells (SCLCCs) are a subpopulation of tumor cells thought to be responsible for treatment resistance, development of metastases and tumor recurrence, making the targeting of this cell population critical. We sought to evaluate the effect of lerociclib, a CDK4/6 inhibitor, on pediatric sarcoma cell stemness. Methods: We investigated two established human osteosarcoma cells lines (U2-OS and MG-63), two metastatic synovial sarcoma patient-derived xenografts (PDXs) (COA-30 and COA-79) and a metastatic epithelioid sarcoma PDX (COA-171). Cells were treated with the CDK4/6 inhibitor, lerociclib, at concentrations below the known LD50. Synovial sarcoma stemness markers Octamer-binding transcription factor 4 (Oct4), homeobox protein Nanog, SOX 2 and nestin were evaluated by qPCR. CD117, a marker of stemness in osteosarcoma cells, was examined by flow cytometry. Finally, long-term passaged U2-OS and MG-63 cells were placed in low attachment serum-free conditions, and tumorsphere formation was evaluated using extreme limiting dilution assay (ELDA) in all cell lines. Results: Lerociclib treatment significantly decreased abundance of Oct4 (by 54%), Nanog (by 29%), SOX 2 (by 60%) and nestin (by 63%) mRNA in COA-30 synovial sarcoma cells. Cell surface expression of CD117 decreased from 17% to 11% in MG-63 cells when treated with lerociclib 2μM. Treatment of all cell types with lerociclib led to significantly decreased tumorsphere formation (Table). Conclusions: Treatment with lerociclib led to a decrease in mRNA abundance in known synovial sarcoma stem cell markers, a decrease in CD117 cell surface expression in osteosarcoma cells and decreased the ability of cells to form tumorspheres. These findings indicate lerociclib leads to decreased sarcoma cell stemness, which plays a key role in tumor progression and recurrence and should be further investigated for potential translation to the clinical setting.[Table: see text]

Mechanisms during Osteogenic Differentiation in Human Dental Follicle Cells.

Human dental follicle cells (DFCs) as periodontal progenitor cells are used for studies and research in regenerative medicine and not only in dentistry. Even if innovative regenerative therapies in medicine are often considered the main research area for dental stem cells, these cells are also very useful in basic research and here, for example, for the elucidation of molecular processes in the differentiation into mineralizing cells. This article summarizes the molecular mechanisms driving osteogenic differentiation of DFCs. The positive feedback loop of bone morphogenetic protein (BMP) 2 and homeobox protein DLX3 and a signaling pathway associated with protein kinase B (AKT) and protein kinase C (PKC) are presented and further insights related to other signaling pathways such as the WNT signaling pathway are explained. Subsequently, some works are presented that have investigated epigenetic modifications and non-coding ncRNAs and their connection with the osteogenic differentiation of DFCs. In addition, studies are presented that have shown the influence of extracellular matrix molecules or fundamental biological processes such as cellular senescence on osteogenic differentiation. The putative role of factors associated with inflammatory processes, such as interleukin 8, in osteogenic differentiation is also briefly discussed. This article summarizes the most important insights into the mechanisms of osteogenic differentiation in DFCs and is intended to be a small help in the direction of new research projects in this area.

The transcription factors TLR1 and TLR2 negatively regulate trichome density and artemisinin levels in Artemisia annua.

The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density; however, the factors affecting trichome development are largely unknown. Here, we demonstrate that the A. annua R2R3 MYB transcription factor TrichomeLess Regulator 1 (TLR1) negatively regulates trichome development. In A. annua, TLR1 overexpression lines had 44.7%-64.0% lower trichome density and 11.5%-49.4% lower artemisinin contents and TLR1-RNAi lines had 33%-93.3% higher trichome density and 32.2%-84.0% higher artemisinin contents compared with non-transgenic controls. TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A. annua. When heterologously expressed in Arabidopsis thaliana, TLR1 interacts with GLABROUS3a, positive regulator of trichome development, and represses trichome development. Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox (WOX) protein AaWOX1, which interacts with the LEAFY-like transcription factor TLR2. TLR2 overexpression in Arabidopsis and A. annua showed that TLR2 reduces trichome development by reducing gibberellin levels. Furthermore, artemisinin contents were 19%-43% lower in TLR2-overexpressing A. annua plants compared to controls. These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.

MO425: Rosuvastatin Activates Hox13-Usag-1 Pathway and Prevents Renal Fibrosis

Abstract BACKGROUND AND AIMS Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, are primarily cholesterol-lowering drugs that have become standard of care in the primary and secondary prevention of cardiovascular diseases. Apart from lipid-lowering, statins may act beneficially through anti-fibrotic mechanisms to protect the diseased kidney. This study evaluated anti-fibrotic effects of rosuvastatin (RSV) in a chronic kidney fibrosis model and against the TGF-ß1 stimulated Madin-Darby canine kidney (MDCK) cells in vitro. METHOD Mice subjected to unilateral ischemic reperfusion injury with contralateral nephrectomy (uIRIx) were treated with vehicle or RSV (10 mg/kg, by oral gavage) daily for 4 weeks and kidneys were analyzed for markers of fibrosis, bone morphogenetic protein-7 (BMP-7), uterine sensitization-associated gene-1 (USAG-1), and SMAD signaling. Control and homeobox protein Hox-A13 (HOXA13) knocked down MDCK cells were stimulated with TGF- ß1 (5 ng/ml) and then treated with RSV. RESULTS Kidneys from uIRIx mice showed increased expression of α-SMA, Collagen 1 and decrease in BMP-7 (20.35 ± 2.37 versus 1.00 ± 0.27, P &amp;lt; 0.05; 8.43 ± 1.55 versus 1.00 ± 0.35, P &amp;lt; 0.05; 0.75 ± 0.06 versus 1.00 ± 0.15, P &amp;lt; 0.05, respectively). In contrast, expression of USAG-1, a BMP-7 antagonist, was markedly increased in fibrotic kidney (6.60 ± 1.11 versus 1.00 ± 0.02, P &amp;lt; 0.05). Interestingly, RSV treatment not only attenuated expression of USAG-1 (0.72 ± 0.08 versus 1.36 ± 0.14, P &amp;lt; 0.05) but also showed a tendency to activate expression of HOXA13 (0.36 ± 0.14 versus 0.51 ± 0.15, P &amp;gt; 0.05) and improved other markers of fibrosis. Moreover, RSV treatment significantly reduced phosphorylated Smad3 (3.94 ± 0.81 versus 7.17 ± 1.50, P &amp;lt; 0.05) and increased phosphorylation levels of Smad 1/5/9 (0.67 ± 0.10 versus 0.30 ± 0.09, P &amp;lt; 0.05) that is associated with BMP-7 signaling in the fibrotic kidney. MDCK cells stimulated with TGF-β1 in vitro showed increased expression of α-SMA, fibronectin, vimentin, collagen 1, USAG-1, and phosphorylation of Smad3 as well as decreased expression of phosphorylated Smad 1/5/9. RSV treatment significantly reversed these changes as well as increased level of transcriptional factor HOXA13 (0.89 ± 0.12 versus 0.33 ± 0.08, P &amp;lt; 0.05), which negatively regulates USAG-1, without changes in BMP-7 expression. In addition, effect of RSV treatment on USAG-1 expression was significantly decreased in HOXA13 gene knocked down MDCK cells (1.01 ± 0.19 versus 2.04 ± 0.38, P &amp;lt; 0.05; 1.66 ± 0.13 versus 2.04 ± 0.38, P &amp;gt; 0.05; TGF- ß1 + RSV versus TGF- ß1 and TGF- ß1 + RSV + siRNA vs TGF- ß1, respectively). These in vitro data suggest that RSV enhanced anti-fibrotic pathway by downregulating a dominant BMP-7 antagonist USAG-1 via HOXA13 upregulation and not by enhancing BMP-7 production. CONCLUSION The present results demonstrate that RSV inhibits the progression of kidney fibrosis in part by upregulating BMP-7-mediated signaling via HOXA13 expression and down regulation of USAG-1.

OTX2's Dual Mode of Degradation is Regulated by O‐GlcNAcylation

The O‐GlcNAc post‐translational modification is reversibly added onto intracellular proteins. It is a unique glucose rheostat for cell signaling relying on the availability of UDP‐GlcNAc, itself reflecting extracellular glucose. With more than 7000 human targets identified to date, O‐GlcNAcylation regulates numerous physiological processes such as cell cycle, transcriptional/translational regulation, protein localization or degradation, and development. Therefore, O‐GlcNAcylation is a molecular bridge between dietary glucose level and proper signaling regulation.Using cellular and mouse models, our lab has previously delved into the consequences of hyper‐O‐GlcNAcylation in the brain. Among phenotypes of obesity and growth defects, the pituitary gland of these mice was generally delayed in development. A critical aspect of pituitary’s ontogeny is the transient expression of the homeobox protein OTX2, an O‐GlcNAcylated protein. However, the function of O‐GlcNAcylation in regulating OTX2 has not been investigated. Interestingly, in hyper‐O‐GlcNAcylated mouse embryonic stem cells, this transcription factor’s expression increased, suggesting that O‐GlcNAc plays a role in OTX2 stability/degradation. Thus, we hypothesized that OTX2 is degraded by the proteasome and O‐GlcNAc cycling regulates its timely degradation.Using a combination of proteasome inhibition (MG‐132 mediated) and O‐GlcNAc increase (Thiamet‐G, TG), we demonstrated that endogenous OTX2 was indeed degraded by the proteasome. Interestingly, increased O‐GlcNAc levels caused further stabilization of OTX2, additional to the one achieved with proteasome inhibition alone. This suggested that OTX2 utilizes another degradation pathway, possibly autophagy. We showed that the macroautophagy inhibitor Chloroquine (CQ) prevented OTX2 degradation, and the addition of TG did not further stabilize the protein. This confirmed that OTX2 has two degradation modes, and that O‐GlcNAcylation is involved in the autophagy‐mediated degradation of this protein. However, we believe that autophagic degradation of OTX2 only occurs when this homeobox protein is abnormally overexpressed, such as in Medulloblastoma. Similar crosstalk between proteasome and autophagy has been demonstrated by others for developmental transcription factors like SOX2 and OCT4, following proteasome inhibition. We performed mass spectrometry site‐mapping of OTX2, and identified three O‐GlcNAc sites in the central domain of OTX2, likely involved in its autophagy‐mediated degradation.To summarize, this study highlights the O‐GlcNAc modification as a nutrient‐dependent sensor that regulates the homeobox protein OTX2 during pituitary and brain development. Like many homeobox proteins, OTX2 level needs to be tightly regulated for proper patterning and development, and its deregulation amongst other proteins is a major driver of Medulloblastoma. Therefore, we foresee that O‐GlcNAcylated OTX2 may play a major role in Medulloblastoma pathogenesis.