Msh Homeobox Research Articles

Msx genes delineate a novel molecular map of the developing cerebellar neuroepithelium.

In the early cerebellar primordium, there are two progenitor zones, the ventricular zone (VZ) residing atop the IVth ventricle and the rhombic lip (RL) at the lateral edges of the developing cerebellum. These zones give rise to the several cell types that form the GABAergic and glutamatergic populations of the adult cerebellum, respectively. Recently, an understanding of the molecular compartmentation of these zones has emerged. To add to this knowledge base, we report on the Msx genes, a family of three transcription factors, that are expressed downstream of Bone Morphogenetic Protein (BMP) signaling in these zones. Using fluorescent RNA in situ hybridization, we have characterized the Msx (Msh Homeobox) genes and demonstrated that their spatiotemporal pattern segregates specific regions within the progenitor zones. Msx1 and Msx2 are compartmentalized within the rhombic lip (RL), while Msx3 is localized within the ventricular zone (VZ). The relationship of the Msx genes with an early marker of the glutamatergic lineage, Atoh1, was examined in Atoh1-null mice and it was found that the expression of Msx genes persisted. Importantly, the spatial expression of Msx1 and Msx3 altered in response to the elimination of Atoh1. These results point to the Msx genes as novel early markers of cerebellar progenitor zones and more importantly to an updated view of the molecular parcellation of the RL with respect to the canonical marker of the RL, Atoh1.

SP6 controls human cytotrophoblast fate decisions and trophoblast stem cell establishment by targeting MSX2 regulatory elements

The commitment and differentiation of human placental progenitor cytotrophoblast (CT) cells are crucial for a successful pregnancy, but the underlying mechanism remains poorly understood. Here, we identified the transcription factor (TF), specificity protein 6 (SP6), as a human species-specific trophoblast lineage TF expressed in human placental CT cells. Using pluripotent stem cells as a model, we demonstrated that SP6 controls CT generation and the establishment of trophoblast stem cells (TSCs) and identified msh homeobox 2 (MSX2) as the downstream effector in these events. Mechanistically, we showed that SP6 interacts with histone acetyltransferase P300 to alter the landscape of H3K27ac at targeted regulatory elements, thereby favoring transcriptional activation and facilitating CT cell fate decisions and TSC maintenance. Our results established SP6 as a regulator of the human trophoblast lineage and implied its role in placental development and the pathogenies of placental diseases.

Bilateral Foramina Parietalia Permagna - A Calvarial Defect Caused by Haploinsufficiency of the Msh Homeobox 2 Gene: A Case Report and Current Literature Review.

Foramina parietalia permagna (FPP) is a rare anatomical defect that affects the parietal bones of the human skull. FPP is characterized by symmetric perforations on either side of the skull, which are caused by insufficient ossification during embryogenesis. These openings are typically abnormally large and can range from a few millimeters to several centimeters in diameter. Enlarged foramina are often discovered incidentally during anatomical or radiological examinations and in most cases left untreated unless symptoms develop. Although this calvarial defect is usually asymptomatic, it may be accompanied by neurological or vascular conditions that can have clinical significance in certain cases. FPP is an inherited disorder and arises due to mutations in either Msh homeobox 2 (MSX2) or aristaless-like homeobox 4 (ALX4) genes. In almost all cases, one parent is affected. Clinical findings and diagnostic imaging typically contribute to determine the diagnosis.

MiR-21-5p-loaded bone mesenchymal stem cell-derived exosomes repair ovarian function in autoimmune premature ovarian insufficiency by targeting MSX1

What is the effect of micro-RNA (miR)-21-5p-loaded bone marrow mesenchymal stem cell-derived exosomes (miR-21-Exo) on autoimmune premature ovarian insufficiency (POI)? The Cell Counting Kit 8 (CCK8) assay, fluorescence-activated cell sorting, western blotting, quantitative reverse transcriptase (qRT)-PCR and enzyme-linked immunosorbent assay (ELISA) verified the effect of miR-21-Exo on interferon-γ (IFN-γ)-induced KGN cells. qRT-PCR, western blotting and dual-luciferase reporter gene assays verified that miR-21-Exo mediated Msh homeobox 1 (MSX1) regulation of the Notch signalling pathway and that miR-21 interacted directly with MSX1. The effects of miR-21-Exo on the ovaries were verified by monitoring of the oestrous cycle, haematoxylin and eosin staining, follicle counts, ELISA, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), western blotting and qRT-PCR. The results showed that miR-21-Exo promoted IFN-γ-induced KGN cell proliferation and hormone synthesis, and inhibited apoptosis. Using dual-luciferase reporter gene assays, miR-21 and MSX1 were shown to have direct interactions. Moreover, the findings elucidated that miR-21-Exo inhibited cell apoptosis and promoted hormone synthesis by mediating MSX1 to regulate the Notch signalling pathway. miR-21-Exo restored the ovarian structure in a mouse model of autoimmune POI, promoted endocrine function and proliferation, and inhibited apoptosis and inflammation in vivo. This study demonstrates that miR-21-Exo regulates the MSX1-mediated Notch signalling pathway to inhibit granulosa cell apoptosis and improve hormone synthesis function, providing insight into a potential mechanism of molecular therapy for the treatment of autoimmune POI.

The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity

ABSTRACT The mammalian endometrium is covered by the lumenal epithelium (Le), which directly interacts with the blastocyst and plays an important role in the establishment of reciprocal crosstalk between the embryo and receptive uterus during implantation. However, the effect of the blastocyst on uterine differentiation during the window of receptivity is far from well understood. Through transcriptomic profiling of the uterine Le isolated by laser capture microdissection (LCM), it was demonstrated that global gene expression changes occurred in Le between pseudopregnant mice without embryos and pregnant mice with embryos. Some differentially expressed genes, including upregulated Areg (amphiregulin), Ihh (Indian hedgehog), Lifr (leukemia inhibitory factor receptor) and downregulated Msx1 (msh homeobox 1), Pgr (progesterone receptor), and Gata2 (GATA binding protein 2) in pregnant mice, have been reported to regulate the establishment of uterine receptivity. Besides, we found that blastocysts induced an increase in both the number and acidification of lysosome, consistent with enhanced lysosomal hydrolase activity in uterine Le. Further exploration uncovered that blastocyst-derived IGF2 was involved into the activation of epithelial STAT3 to induce lysosomal hydrolase expression, and inhibition of lysosomal function derails both uterine receptive maker gene expressions and embryo implantation. Finally, based on the proteomic data of both epithelia and the separated lysosome, it was revealed that CLDN1 (claudin 1) and MUC1 (mucin 1, transmembrane), two well-known downregulated molecules for successful implantation, are degraded by epithelial lysosome. In brief, our data demonstrated that blastocysts induced normal epithelium differentiation with lysosome activation to promote the uterine epithelial differentiation for embryo implantation. Abbreviations ACTB: actin beta; AREG: amphiregulin; ATP6V0A4: ATPase, H+ transporting, lysosomal V0 subunit A4; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CLDN1: claudin 1; CTSB: cathepsin B; DEGs: differentially expressed genes; E2: 17β-estradiol; ESR: estrogen receptor; GATA2: GATA binding protein 2; GLA: galactosidase, alpha; GO: gene ontology; HBEGF: heparin-binding EGF-like growth factor; IGF1R: insulin-like growth factor 1 receptor; Ihh: Indian hedgehog; ISH: in situ hybridization; LAMP1: lysosomal-associated membrane protein 1; LCM: laser capture microdissection; Le: lumenal epithelium; LGMN: legumain; LIF: leukemia inhibitory factor; LIFR: LIF receptor alpha; MSX1: msh homeobox 1; MUC1: mucin 1, transmembrane; P4: progesterone; PBS: phosphate-buffered saline; PCA: principal component analysis; PPT1: palmitoyl-protein thioesterase 1; PGR: progesterone receptor; PSP: pseudopregnancy; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; qPCR: quantitative real-time polymerase chain reaction; SP: pregnancy; TFEB: transcription factor EB.

Measurement and analysis of the crown conical degree of maxillary incisors in patients with congenital tooth agenesis caused by different gene mutations

Objective: To measure the crown conical degree of the remaining maxillary incisors in patients with congenital tooth agenesis, and to analyze the influence of different gene mutations on the crown conical degree of patients. Methods: Whole exome sequencing was performed on 85 patients with congenital tooth agenesis (50 males, 35 females, median age 19 years old) who visited the Department of Prosthodontics, Peking University School and Hospital of Stomatology from January 2019 to January 2023. The pathogenic gene was identified. The width of the crowns of the maxillary central and lateral incisors at the incisal 1/3 and gingival 1/3 were measured on the panoramic radiograph, and the ratio was defined as the crown conical degree. The smaller the ratio is, the more likely is the crown to be peg shaped teeth. The control group was matched by age and gender with 85 other patients with intact maxillary permanent incisors who were treated in the Department of Prosthodontics, Peking University School and Hospital of Stomatology from January 2019 to January 2023. The conical degree of the tooth agenesis group was compared with the control group by t-test, and the differences in the crown conical degree in different gene mutation groups were compared using one-way analysis of variance, and the multiple comparisons among gene groups were carried out using the LSD method. Results: Among the 85 tooth agenesis patients, the numbers of patients in each gene mutation group were 20 in ectodysplasin A (EDA) group, 8 in ectodysplasin A receptor (EDAR) group, 15 in wingless-type MMTV integration site family, member 10A (WNT10A) group, 16 in paired box 9 (PAX9) group, 10 in Msh homeobox 1 (MSX1) group, 10 in low-density Lipoprotein receptor related protein 6 (LRP6) group, and 6 in bone morphogenetic protein4 (BMP4) group. The number of missing teeth were 1-27, median number 15 among the tooth agenesis patients. There was no significant difference in the conical degree between the left and right homonymous teeth in the congenital tooth agenesis group and the control group (P>0.05). The crown conical degree of maxillary central incisor and lateral incisor in the congenital missing teeth group (0.95±0.24, 0.90±0.22) was significantly smaller than that in the control group (1.12±0.09, 1.13±0.09) (t=-8.50, P<0.001; t=-11.47, P<0.001). In WNT10A mutants, the conical degree of lateral incisors (0.89±0.18) was less than that of central incisors (1.07±0.15)(t=3.68, P<0.001). The conical degree of central incisors and lateral incisors (0.70±0.23, 0.57±0.15) of EDA mutants was significantly lower than that in patients with other gene mutations (P>0.05). Conclusions: Compared with the normal control group, the remaining maxillary central and lateral incisors of the seven gene mutation groups of patients with congenital tooth agenesis all had different degrees of conical crown. Among them, the crown conical degree of maxillary central and lateral incisors of the EDA mutation was the most severe, and the WNT10A mutation affected the maxillary lateral incisors more specifically.

#3986 THE ROLE AND MECHANISM OF EZH2 IN VASCULAR CALCIFICATION ASSOCIATED WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Vascular calcification (VC) is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. EZH2 (Enhancer of zeste 2 homolog-2) is reported as a key epigenetic regulator involved in various kidney diseases such as acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. In this study, we aimed to investigated the role of EZH2 in chronic kidney disease associated vascular calcification. Method 1. Patients and Radial Artery Analysis Radial arteries with or without calcification from patients with end-stage renal disease that underwent arterial venous fistular surgery were collected for Von Kossa staining and immunohistochemical (IHC) staining. 2. Animal model of Chronic renal failure-Vascular Calcification (CRF-VC) (in vivo) An adenine and phosphate (1.2%) diet-induced CRF mouse model was designed following an 8-week program. The thoracoabdominal arteries of mice and rats were dissected to assay Ca deposition and histological analysis. Plasma levels of Alkaline phosphate (ALP), and phosphate (Pi) were measured. The transcription level of EZH2 and Runt-related transcription factor 2 (Runx2) were measured by real-time PCR and IHC staining. 3. Aortic ring calcification (ex vivo) Thoracic aortas were dissected from SD rat. To induce calcification, Pi (NaH2PO4/Na2HPO4 [pH = 7.4]) was added to HG-DMEM at a final concentration of 2.6 mmol/l. After the indicated incubation periods (3, 5, 7 days), samples were taken for Calcium deposition assay, Western blotting and histological analysis. 4. Cell culture and treatment (in vitro) Calcification of rat smooth muscle embryonic thoracic aorta cell line A7r5 (VSMC) was induced by incubation in calcifying media. 3-deazaneplanocin A (3-DZNeP), a carbocyclic analog of adenosine was added right after Pi to block EZH2 activation. After the indicated incubation periods, samples were collected for Calcium deposition assay and Western blotting analysis. Results 1. In vivo, EZH2 protein level was increased in calcified radial arteries from patients with end-stage renal disease that underwent arterial venous fistular operation. In CRF-VC mice model, the aortic calcium deposition markedly aggravated in the group induced by adenine diet. Severe vascular calcification was also induced as shown by increased intensity of von Kossa. Moreover, IHC and q-PCR results showed that the protein and transcriptional levels of EZH2 expression were upregulated in calcified aortas of CRF mice, in paralleled with the increased expression Runx2 in protein and transcriptional levels. 2. Ex vivo, histological analysis revealed significant medial vascular calcification in the aortic ring culture with this calcifying medium for 7 days. Similarly, WB results showed that the expression of EZH2 and its downstream, methylation of Histone H3 at lysine 27 (H3K27me3), were significantly enhanced after high Pi medium stimulation while the osteogenic markers Runx2, Osteopontin (OPN) and Msh homeobox 2 (Msx2) protein levels were upregulated and the expression of VSMC differentiation markers α-Smooth muscle actin (α-SMA), Calponin (CNN) and Smooth muscle protein 22 (SM22) were downregulated. 3. In vitro, the osteogenic markers Runx2, OPN and Msx2 were significantly upregulated in high Pi treated VSMCs compared to control VSMCs. Moreover, the epigenetic markers EZH2 and H3K27me3a were markedly repressed in calcifying VSMCs by time. 4. 3-DZNep treatment reduced the calcium deposition in a concentration dependent manner. Western blotting showed that 3-DZNep treatment blocked the increased expression of osteogenic markers Runx2, Bone morphogenetic protein 2 (BMP2) as well as epigenetic markers EZH2 and H3K27me3a by high Pi treatment, indicating that inhibition of EZH2 attenuates VSMCs Calcification. Conclusion Our study revealed that EZH2 is highly expressed in calcified vascular tissues of CRF patients and CRF mice, rat aorta culture and VSMCs calcification models, and EZH2 inhibitor 3-DZNep attenuated calcification of VSMCs. EZH2 could be a promising target for treatment of vascular calcification in CRF patients.

Influence of FOSL1 Inhibition on Vascular Calcification and ROS Generation through Ferroptosis via P53-SLC7A11 Axis.

Vascular calcification during aging is highly prevalent in patients with cardiovascular disease; however, there is still no improvement in clarifying the development of vascular calcification. FOSL1 is a transcription regulator belonging to the AP-1 family, which has a unique function in vascular senescence, but its role in vascular calcification needs to be further explored. Primary mouse vascular smooth muscle cells were isolated and used to construct a calcification model in vitro. Seven-week-old male C57BL/6 mice were used to build the vitD3-induced calcification model in vivo. qRT-PCR and western blot were used to verify the expression of FOSL1 and other genes expressed in vascular smooth muscle cells and aortas. The level of calcification was determined by Alizarin Red S (ARS) staining and the calcium content assay. The level of cellular GSH was detected by the GSH assay kit. Here, we report that FOSL1 was up-regulated after high-calcium/phosphate treatment in both the in vivo and in vitro vascular calcification models. Functional studies have shown that the reduction of FOSL1 attenuates ferroptosis and calcification in vascular smooth muscle cells, as indicated by ARS staining, calcium content assay, and western blot. The inhibition of FOSL1 downregulated the expression of bone-related molecules including Msh Homeobox 2 (MSX2) and tumor necrosis factor receptor superfamily, member 11b/osteoprotegerin (OPG), suggesting that FOSL1 promoted osteogenic differentiation of vascular smooth muscle cells. Furthermore, we found that the ferroptosis-inducing drug erastin can significantly accelerate calcification in the aortic ring while Ferrostatin-1 (fer-1), a drug to protect cells from ferroptosis, can alleviate calcification. Further experiments have shown that inhibiting FOSL1 can promote the expression of ferroptosis-related genes and attenuate calcification. Functionally, cellular GSH levels were increased after the reduction of FOSL1. In this study, we observed a significant protective effect when we reduced the expression of FOSL1 during vascular calcification, and this effect might regulate ferroptosis to a great extent.

CLU (clusterin) promotes mitophagic degradation of MSX2 through an AKT-DNM1L/Drp1 axis to maintain SOX2-mediated stemness in oral cancer stem cells

ABSTRACT Mitophagy regulates cancer stem cell (CSC) populations affecting tumorigenicity and malignancy in various cancer types. Here, we report that cisplatin treatment led to the activation of higher mitophagy through regulating CLU (clusterin) levels in oral CSCs. Moreover, both the gain-of-function and loss-of-function of CLU indicated its mitophagy-specific role in clearing damaged mitochondria. CLU also regulates mitochondrial fission by activating the Ser/Thr kinase AKT, which triggered phosphorylation of DNM1L/Drp1 at the serine 616 residue initiating mitochondrial fission. More importantly, we also demonstrated that CLU-mediated mitophagy positively regulates oral CSCs through mitophagic degradation of MSX2 (msh homeobox 2), preventing its nuclear translocation from suppressing SOX2 activity and subsequent inhibition of cancer stemness and self-renewal ability. However, CLU knockdown disturbed mitochondrial metabolism generating excessive mitochondrial superoxide, which improves the sensitivity to cisplatin in oral CSCs. Notably, our results showed that CLU-mediated cytoprotection relies on SOX2 expression. SOX2 inhibition through genetic (shSOX2) and pharmacological (KRX-0401) strategies reverses CLU-mediated cytoprotection, sensitizing oral CSCs toward cisplatin-mediated cell death.

Dynamic expression of Mage-D1 in rat dental germs and potential role in mineralization of ectomesenchymal stem cells

Mage-D1 (MAGE family member D1) is involved in a variety of cell biological effects. Recent studies have shown that Mage-D1 is closely related to tooth development, but its specific regulatory mechanism is unclear. The purpose of this study was to investigate the expression pattern of Mage-D1 in rat dental germ development and its differential mineralization ability to ectomesenchymal stem cells (EMSCs), and to explore its potential mechanism. Results showed that the expression of Mage-D1 during rat dental germ development was temporally and spatially specific. Mage-D1 promotes the proliferation ability of EMSCs but inhibits their migration ability. Under induction by mineralized culture medium, Mage-D1 promotes osteogenesis and tooth-forming ability. Furthermore, the expression pattern of Mage-D1 at E19.5 d rat dental germ is similar to p75 neurotrophin receptor (p75NTR), distal-less homeobox 1 (Dlx1) and msh homeobox 1 (Msx1). In addition, Mage-D1 is binding to p75NTR, Dlx1, and Msx1 in vitro. These findings indicate that Mage-D1 is play an important regulatory role in normal mineralization of teeth. p75NTR, Dlx1, and Msx1 seem to be closely related to the underlying mechanism of Mage-D1 action.

Regulation of feather follicle development and Msx2 gene SNP degradation in Hungarian white goose

BackgroundHungarian white goose has excellent down production performance and was introduced to China in 2010. The growth and development of feather follicles has an important impact on down production. Goose feather follicles can be divided into primary and secondary feather follicles, both of which originate in the embryonic stage. Msx2 (Msh Homeobox 2) plays a regulatory role in tissues and organs such as eyes, teeth, bones and skin. However, its regulatory mechanism on goose feather follicles development remains unclear.ResultsMsx2 gene first increased, then decreased and increased at the end (E13, E18, E23, E28) during embryonic feather follicle development, and the expression level was the highest at E18. The pEGFP-N1-Msx2 overexpression vector and si-Msx2 siRNA vector were constructed to transfect goose embryo dermal fibroblasts. The results showed that the cell viability of ov-Msx2 group was significantly increased, and the gene expression levels of FGF5 and TGF-β1 genes were significantly down-regulated (P < 0.05), the expressions of PCNA, Bcl2, CDK1, FOXN1 and KGF genes were significantly up-regulated (P < 0.05). After transfection of siRNA vector, the cell viability of the si-Msx2 group was significantly decreased (P < 0.01) compared with the si-NC group. TGF-β1 expression was significantly up-regulated (P < 0.05), FGF5 expression was extremely significantly up-regulated (P < 0.01), while PCNA, Bcl2, CDK1, FOXN1 and KGF gene expression was significantly down-regulated (P < 0.05). High-throughput sequencing technology was used to mine the exon SNPs of Msx2. A total of 11 SNP loci were screened, four of the SNPs located in exon 1 were missense mutations. The feather follicle diameter of the GC genotype at the G78C site is significantly larger than that of the other two genotypes.ConclusionsMsx2 maybe inhibit the apoptosis of goose dermal fibroblasts and promotes their proliferation. G78C can be used as a potential molecular marker for downy Variety.

Attenuating effect of magnesium on pulmonary arterial calcification in rodent models of pulmonary hypertension.

Vascular calcification has been considered as a potential therapeutic target in pulmonary hypertension. Mg2+ has a protective role against calcification. This study aimed to investigate whether Mg2+ could alleviate pulmonary hypertension by reducing medial calcification of pulmonary arteries. Monocrotaline (MCT)-induced and chronic hypoxia-induced pulmonary hypertension rats were given an oral administration of 10% MgSO4 (10 ml/kg per day). Additionally, we administered Mg2+ in calcified pulmonary artery smooth muscle cells (PASMCs) after incubating with β-glycerophosphate (β-GP, 10 mmol/l). In vivo, MCT-induced and chronic hypoxia-induced pulmonary hypertension indexes, including right ventricular systolic pressure, right ventricular mass index, and arterial wall thickness, as well as Alizarin Red S (ARS) staining-visualized calcium deposition, high calcium levels, and osteochondrogenic differentiation in pulmonary arteries, were mitigated by dietary Mg2+ intake. In vitro, β-GP-induced calcium-rich deposits stained by ARS, calcium content, as well as the detrimental effects of calcification to proliferation, migration, and resistance to apoptosis of PASMCs were alleviated by high Mg2+ but exacerbated by low Mg2+. Expression levels of mRNA and protein of β-GP-induced osteochondrogenic markers, RUNX Family Transcription Factor 2, and Msh Homeobox 2 were decreased by high Mg2+ but increased by low Mg2+; however, Mg2+ did not affect β-GP-induced expression of SRY-Box Transcription Factor 9. Moreover, mRNA expression and protein levels of β-GP-reduced calcification inhibitor, Matrix GLA protein was increased by high Mg2+ but decreased by low Mg2+. Mg2+ supplement is a powerful strategy to treat pulmonary hypertension by mitigating pulmonary arterial calcification as the calcification triggered physiological and pathological changes to PASMCs.

Effect of LncRNA-MALAT1 on mineralization of dental pulp cells in a high-glucose microenvironment.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) belongs to the long non-coding RNA (LncRNA) family. LncRNA-MALAT1 is expressed in a variety of tissues and is involved in a variety of diseases and biological processes. Although LncRNA-MALAT1 is upregulated in a high-glucose microenvironment and may participate in odontogenic differentiation, the underlying mechanism is not yet well elucidated. Here, we show that MALAT1 was mainly expressed in the cytoplasm of dental pulp cells (DPCs) in situ hybridization. In addition, high levels of mineralization-related factors, namely, tumor growth factors β 1 and 2 (TGFβ-1 and TGFβ-2), bone morphogenetic proteins 2 and 4 (BMP2 and BMP4), bone morphogenetic protein receptor 1 (BMPR1), SMAD family member 2 (SMAD2), runt-related transcription factor 2 (RUNX2), Msh homeobox 2 (MSX2), transcription factor SP7 (SP7), alkaline phosphatase (ALP), dentin matrix acidic phosphoprotein 1 (DMP1), and dentin sialophosphoprotein (DSPP), were expressed, and MALAT1 was significantly overexpressed in DPCs 7 and 14 days after mineralization induction in a high-glucose microenvironment, but only TGFβ-1, BMP2, MSX2, SP7, ALP, and DSPP were significantly downregulated in DPCs after MALAT1 inhibition. MALAT1 may participate in the mineralization process of DPCs by regulating multiple factors (TGFβ-1, BMP2, MSX2, SP7, ALP, and DSPP).

Transcriptional regulation of FRZB in chondrocytes by Osterix and Msx2

Osteoarthritis is a common joint disease that causes destruction of articular cartilage and severe inflammation surrounding knee and hip joints. However, to date, effective therapeutic reagents for osteoarthritis have not been developed because the underlying molecular mechanisms are complex. Recent genetic findings suggest that a Wnt antagonist, frizzled-related protein B (FRZB), is a potential therapeutic target for osteoarthritis. Therefore, this study aimed to examine the transcriptional regulation of FRZB in chondrocytes. Frzb/FRZB expression was assessed by RT-qPCR analyses in murine articular chondrocytes and SW1353 chondrocyte cell line. Overexpression and knockdown experiments were performed using adenovirus and lentivirus, respectively. Luciferase-reporter and chromatin immunoprecipitation assays were performed for determining transcriptional regulation. Protein-protein interaction was determined by co-immunoprecipitation analysis. Frzb was highly expressed in cartilages, especially within articular chondrocytes. Interleukin-1α markedly reduced Frzb expression in articular chondrocytes in association with cartilage destruction and increases in ADAM metallopeptidase with thrombospondin type 1 motif (Adamts) 4 and Adamts5 expression. Bone morphogenetic protein 2 (BMP2) increased FRZB expression in SW1353 cells through Smad signaling. Osterix and msh homeobox 2 (Msx2), both of which function as downstream transcription factors of BMP2, induced FRZB expression and upregulated its promoter activity. Co-immunoprecipitation results showed a physical interaction between Osterix and Msx2. Knockdown of either Osterix or Msx2 inhibited BMP2-dependent FRZB expression. Chromatin immunoprecipitation indicated a direct association of Osterix and Msx2 with the FRZB gene promoter. These results suggest that BMP2 regulates FRZB expression through Osterix and Msx2.

Genome-wide screening for deubiquitinase subfamily identifies ubiquitin-specific protease 49 as a novel regulator of odontogenesis

Proteins expressed by the paired box gene 9 (PAX9) and Msh Homeobox 1 (MSX1) are intimately involved in tooth development (odontogenesis). The regulation of PAX9 and MSX1 protein turnover by deubiquitinating enzymes (DUBs) plausibly maintain the required levels of PAX9 and MSX1 during odontogenesis. Herein, we used a loss-of-function CRISPR-Cas9-mediated DUB KO library kit to screen for DUBs that regulate PAX9 and MSX1 protein levels. We identify and demonstrate that USP49 interacts with and deubiquitinates PAX9 and MSX1, thereby extending their protein half-lives. On the other hand, the loss of USP49 reduces the levels of PAX9 and MSX1 proteins, which causes transient retardation of odontogenic differentiation in human dental pulp stem cells and delays the differentiation of human pluripotent stem cells into the neural crest cell lineage. USP49 depletion produced several morphological defects during tooth development, such as reduced dentin growth with shrunken enamel space, and abnormal enamel formation including irregular mineralization. In sum, our results suggest that deubiquitination of PAX9 and MSX1 by USP49 stabilizes their protein levels to facilitate successful odontogenesis.

MiR-140-5p upregulation suppressed β-glycerophosphate-induced vascular smooth muscle cell calcification via targeting TLR4

Background The role and function of microRNA (miRNA, miR)-140-5p in the calcification of vascular smooth muscle cells (VSMCs) have been explored in this study. Methods The calcium nodules formed in transfected and β-glycerophosphate (β-GP)-treated VSMCs were observed using Alizarin Red S staining, and alkaline phosphatase (ALP) activity was determined. VSMC apoptosis was detected with flow cytometry assay. The target gene of miR-140-5p was predicted and confirmed with dual-luciferase reporter assay. Relative expressions of miR-140-5p, toll like receptor 4 (TLR4) and vascular calcification-related proteins (α-smooth muscle actin, α-SMA; Msh Homeobox 2, MSX2; bone morphogenetic protein 2, BMP2; Kruppel-like factor 4, KLF4; Runt-related transcription factor 2, RUNX2) were measured through quantitative real time polymerase chain reaction (qRT-PCR) and western blot. Results MiR-140-5p upregulation reversed the effects of β-GP on downregulating miR-140-5p and α-SMA expressions, enhancing ALP activity, calcium nodule formation and cell apoptosis, and upregulating levels of MSX2, BMP2, KLF4 and RUNX2. TLR4 was the target of miR-140-5p, and offset the effects of miR-140-5p on β-GP-induced VSMCs. Conclusions MiR-140-5p upregulation represses β-GP-induced calcification of VSMCs via targeting TLR4, providing a potential therapeutic method for vascular calcification.

The effect of dexamethasone on uterine receptivity, mediated by the ERK1/2-mTOR pathway, and the implantation window: An experimental study.

Background The role of glucocorticoids in implantation has been demonstrated.Objective This study aimed to evaluate the effect of dexamethasone on endometrial receptivity.Materials and MethodsIn this experimental study, 40 BALB/c female mice aged eight wk old weighing approximately 25.0 1.4 gr were used. The mice were divided into four groups (n = 10/each) of control, dexamethasone (100 μg/kg, intraperitoneal injection), mammalian target of rapamycin (mTOR) inhibitor (PP242) (30 mg/kg, intraperitoneal injection), and dexamethasone and PP242. The endometrial epithelium of the mouse was separated to measure messenger RNA expression of heart and neural crest derivatives-expressed protein 2 (HAND2), Msh homeobox 1 (Msx-1), heparin binding epidermal growth factor (HB-EGF), microRNA (miRNA) Let-7a, miRNA-145 and miRNA-451, using real-time polymerase chain reaction. Also, protein expression of mammalian mTOR and eukaryotic translation initiation factor 4E-binding protein1 (4E-BP1) was measured using western blot.ResultsThe results revealed that the expression of Msx-1, HAND2, HB-EGF, miRNA-451, and miRNA-Let-7a was significantly decreased in the endometrium in the dexamethasone group compared to the control, while the expression of miRNA-145 in the endometrium was up-regulated. Additionally, the administration of PP242, known as an inhibitor of mTOR, was associated with significantly reduced expression of Msx-1, HAND2, HB-EGF, miRNA-451, and miRNA-Let-7a, while PP242 induced messenger RNA expression of miRNA-145.ConclusionIt appears that dexamethasone can diminish uterine receptivity during the implantation period, at least to some extent, through the alteration of particular genes that impact endometrial receptivity. Furthermore, the mTOR pathway seemingly showed an essential role in endometrial receptivity.

Osteomodulin Gene Expression Is Associated With Plaque Calcification, Stability, and Fewer Cardiovascular Events in the CPIP Cohort.

Stable atherosclerotic plaques are characterized by thick fibrous caps of smooth muscle cells, collagen, and macrocalcifications. Identifying factors of plaque stability is necessary to design drugs to prevent plaque rupture and symptoms. Osteomodulin, originally identified in bones, is expressed by bone synthesizing osteoblasts and involved in mineralization. In the present study, we analyzed osteomodulin expression in human carotid plaques, its link with plaque phenotype, calcification, and future cardiovascular events. Osteomodulin gene expression (OMD; n=82) was determined by RNA sequencing and osteomodulin protein levels by immunohistochemistry (n=45) in carotid plaques obtained by endarterectomy from patients with or without cerebrovascular symptoms from the CPIP (Carotid Plaque Imaging Project) cohort, Skåne University Hospital, Sweden. Plaque components were assessed by immunohistochemistry, RNA sequencing, and multiplex analysis. Patients were followed for cardiovascular events or cardiovascular death during a median of 57 or 70 months, respectively, using national registers. OMD levels were increased in plaques from asymptomatic patients compared to symptomatics. High OMD levels were associated with fewer cardiovascular events during follow-up. OMD correlated positively with smooth muscle α-actin (ACTA2; r=0.73, P=10-13) and collagen (COL1A2; r=0.4, P=0.0002), but inversely with CD68 gene expression (r=-0.67, P=10-11), lipids (r=-0.37, P=0.001), intraplaque hemorrhage (r=-0.32, P=0.010), inflammatory cytokine, and matrix metalloproteinase plaque contents. OMD was positively associated with MSX2 (Msh Homeobox 2) (r=0.32, P=0.003), a marker of preosteoblast differentiation, BMP4 (bone morphogenetic protein) (r=0.50, P=0.000002) and BMP6 (r=0.47, P=0.000007), plaque calcification (r=0.35, P=0.016), and was strongly upregulated in osteogenically stimulated smooth muscle cells, which was further increased upon BMP stimulation. Osteomodulin protein was present in calcified regions. Osteomodulin protein levels were associated with plaque calcification (r=0.41, P=0.006) and increased in macrocalcified plaques. These data show that osteomodulin mRNA and protein levels are associated with plaque calcification in human atherosclerosis. Furthermore, osteomodulin mRNA, but not protein levels, is associated with plaque stability.

Operation of the Atypical Canonical Bone Morphogenetic Protein Signaling Pathway During Early Human Odontogenesis.

Bone morphogenetic protein (BMP) signaling plays essential roles in the regulation of early tooth development. It is well acknowledged that extracellular BMP ligands bind to the type I and type II transmembrane serine/threonine kinase receptor complexes to trigger the BMP signaling pathway. Then, the receptor-activated Smad1/5/8 in cytoplasm binds to Smad4, the central mediator of the canonical BMP signaling pathway, to form transfer complexes for entering the nucleus and regulating target gene expression. However, a recent study revealed the functional operation of a novel BMP-mediated signaling pathway named the atypical BMP canonical signaling pathway in mouse developing tooth, which is Smad1/5/8 dependent but Smad4 independent. In this study, we investigated whether this atypical BMP canonical signaling is conserved in human odontogenesis. We showed that pSMAD1/5/8 is required for the expression of Msh homeobox 1 (MSX1), a well-defined BMP signaling target gene, in human dental mesenchyme, but the typical BMP canonical signaling is in fact not operating in the early human developing tooth, as evidenced by the absence of pSMAD1/5/8-SMAD4 complexes in the dental mesenchyme and translocation of pSMAD1/5/8, and the expression of MSX1 induced by BMP4 is mothers against decapentaplegic homolog 4 (SMAD4)-independent in human dental mesenchymal cells. Moreover, integrative analysis of RNA-Seq data sets comparing the transcriptome profiles of human dental mesenchymal cells with and without SMAD4 knockdown by siRNA displays unchanged expression profiles of pSMAD1/5/8 downstream target genes, further affirming the functional operation of the atypical canonical BMP signaling pathway in a SMAD1/5/8-dependent but SMAD4-independent manner in the dental mesenchyme during early odontogenesis in humans.

Utilizing Shared Big Data to Identify Liver Cancer Dedifferentiation Markers.

Big data reanalysis has the potential to generate novel comparative analyses which aim to generate novel hypotheses and knowledge. However, this approach is underutilized in the realm of cancer research, particularly for cancer stem cells (CSCs). CSCs are a rare subset of tumor cells, which dedifferentiate from healthy adult cells, and have the potential for self-renewal and treatment resistance. This analysis utilizes two publically available single-cell RNA-seq datasets of liver cancer and adult liver cell types to demonstrate how reanalysis of big data can lead to valuable new discoveries. We identify 519 differentially expressed genes between liver CSCs and healthy liver cell types. Here we report the potential novel liver CSC dedifferentiation factor, Msh Homeobox 2, which was significantly upregulated in liver CSCs by 1.36 fold (p-value < 1E-10). These findings have the potential to further advance our knowledge of genes governing the formation of CSCs.