Msh Homeobox Research Articles

Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice

Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox (Msx) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx-overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8, and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx-transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration.

Case Study: Polymelia in a Holstein calf

Polymelia, the manifestation of extra limbs or parts of limbs, occurs when there is a developmental duplication in the novel NHL repeat domain containing protein (NHLRC2) gene in Angus cattle. In Holstein cattle, little is known about the etiology of polymelia. To determine the genetic etiology of polymelia in a Holstein calf, genotyping and sequencing of the Angus developmental duplication locus, karyotyping, and a genome-wide association analysis were performed. The genome-wide association analysis compared the polymelia calf (case) to 2,650 control (normal) Holstein calves using EMMAX-GRM (Efficient Mixed-Model Association eXpedited-Genomic Relationship Matrix) software. In addition, homozygous loci within genes in the case that were never found in a control were also evaluated as putative loci. The polymelia calf had a normal Angus developmental duplication genotype that was confirmed by sequencing. Karyotyping revealed no evidence of chromosomal breakage or gross chromosomal abnormalities in the polymelia calf. The additive genome-wide association analysis identified loci associated with polymelia on BTA13 (P < 1 × 10−281) and BTA10 (P < 2 × 10−110). Seventy-nine candidate genes were identified that contained homozygous genotypes in the case that were never identified in 2,650 controls. Candidate genes include MSH homeobox 2 (MSX2) on BTA20, which has been associated with limb development in mice, and GINS complex subunit 1 (GINS1) on BTA13, which is involved in early embryogenesis in mice. In summary, these results suggest that the etiology of polymelia differs by cattle breed and is the result of genetic variants at more than one locus.

Inhibition of osteo/chondrogenic transformation of vascular smooth muscle cells by MgCl2 via calcium-sensing receptor.

The progression of vascular calcification, an active process promoted by osteo/chondrogenic transformation of vascular smooth muscle cells (VSMCs) is attenuated by activation of the calcium-sensing receptor (CASR). Recent in-vitro studies revealed that vascular calcification could be blunted by Mg, but the underlying mechanisms remained elusive. The present study explored whether the effects of MgCl2 on vascular calcification involve the CASR. Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) and in the mouse vascular calcification model of vitamin D3 overload. Phosphate-induced calcium deposition and mRNA expression of the osteogenic markers msh homeobox 2 (MSX2), CBFA1 (core-binding factor α 1), and ALPL (tissue-nonspecific alkaline phosphatase) in HAoSMCs were blunted by additional treatment with MgCl2. MgCl2 upregulated CASR mRNA expression in HAoSMCs in a dose-dependent manner. Furthermore, the inhibitory effects of MgCl2 on phosphate-induced calcium deposition and osteogenic markers mRNA expression were mimicked by the CASR agonist GdCl3 and reversed by additional treatment with the CASR antagonist NPS-2143 or by silencing of the CASR gene in HAoSMCs. MgCl2 also blunted the osteogenic transformation of VSMCs induced by hydroxyapatite particles. High-dosed cholecalciferol treatment induced vascular calcification and upregulated aortic osteogenic markers Msx2, Cbfa1 and Alpl and collagen type I (Col1a1), collagen type III (Col3a1) and fibronectin (Fbn) mRNA expression in mice, effects reduced by additional treatment with MgCl2. These effects were paralleled by increased aortic Casr mRNA expression in cholecalciferol-treated mice, which was further augmented by MgCl2. The protective effects of MgCl2 on osteo/chondrogenic transformation of VSMCs and vascular calcification involve regulation of CASR and CASR-dependent signaling.

Haplotype-based gene-gene interaction of bone morphogenetic protein 4 and interferon regulatory factor 6 in the etiology of non-syndromic cleft lip with or without cleft palate in a Chilean population.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans, the etiology of which can be dependent on the interactions of multiple genes. We previously reported haplotype associations for polymorphic variants of interferon regulatory factor 6 (IRF6), msh homeobox 1 (MSX1), bone morphogenetic protein 4 (BMP4), and transforming growth factor beta 3 (TGFB3) in Chile. Here, we analyzed the haplotype-based gene-gene interaction for markers of these genes and NSCL/P risk in the Chilean population. We genotyped 15 single nucleoptide polymorphisms (SNPs) in 152 Chilean patients and 164 controls. Linkage disequilibrium (LD) blocks were determined using the Haploview software, and phase reconstruction was performed by the Phase program. Haplotype-based interactions were evaluated using the multifactor dimensionality reduction (MDR) method. We detected two LD blocks composed of two SNPs from BMP4 (Block 1) and three SNPs from IRF6 (Block 2). Although MDR showed no statistical significance for the global interaction model involving these blocks, we found four combinations conferring a statistically significantly increased NSCL/P risk (Block 1-Block 2): T-T/T-G C-G-T/G-A-T; T-T/T-G C-G-C/C-G-C; T-T/T-G G-A-T/G-A-T; and T-T/C-G G-A-T/G-A-T. These findings may reflect the presence of a genomic region containing potential causal variants interacting in the etiology of NSCL/P and may contribute to disentangling the complex etiology of this birth defect.

Kidney stones.

Nature Reviews Disease Primers 2, 16008 (2016) In the version of the article originally published, a typographical error has now been corrected. The statement now reads: Exposure of vascular smooth muscle cells to high levels of calcium and phosphate triggers this transformation154,155, which activates bone morphogenetic proteins (BMPs) and WNT signalling pathways via upregulation of Runt-related transcription factor 2 (RUNX2) and msh homeobox 2 (MSX2) transcription factors156.

Expression of Dlx-5 and Msx-1 in Craniofacial Skeletons and Ilia of Rats Treated With Zoledronate

Because of the different embryologic origins of the craniofacial skeleton and ilium, differences in gene expression patterns have been observed between the jaw bones and ilium. Distal-less homeobox (Dlx) genes and Msh homeobox genes, particularly Dlx-5 and Msx-1, play major roles in cell differentiation and osteogenesis. The purpose of this study was to investigate the effects of zoledronate (ZOL) on the craniofacial skeleton and ilium by detecting changes in Dlx-5 and Msx-1 expression at both the protein and messenger RNA levels. A total of 24 female Sprague-Dawley rats were randomly divided into 2 groups: ZOL group (n= 12), in which the rats were injected intraperitoneally with zoledronic acid for 12weeks, and control group (n= 12), in which the rats were injected with saline solution for 12weeks. By use of immunohistochemistry, Western blotting, and real-time reverse transcription polymerase chain reaction, the expression levels of Dlx-5 and Msx-1 in the craniofacial skeleton (including the maxilla, mandible, and parietal bone) and ilium were examined. Dlx-5 expression in the maxilla and mandible was increased at the protein and messenger RNA levels in the ZOL group compared with the control group (P < .01). In addition, Msx-1 expression in the maxilla and mandible was decreased in the ZOL group (P < .01). Furthermore, Dlx-5 and Msx-1 expression in the ilium was decreased in the ZOL group (P < .05). However, no significant difference in Dlx-5 or Msx-1 expression in the parietal bone was observed between the 2 groups (P > .05). Site-specific differences in the effects of ZOL on the craniofacial skeleton and ilium could be explained by differently altered tendencies in Dlx-5 and Msx-1 expression. The jaw bones were more susceptible to the effects of ZOL than the parietal bone and ilium.

Bcl11b regulates enamel matrix protein expression and dental epithelial cell differentiation during rat tooth development.

Amelogenesis, beginning with thickened epithelial aggregation and ending with highly mineralized enamel formation, is a process mediated by a complex signaling network that involves several molecules, including growth and transcription factors. During early tooth development, the transcription factor B‑cellCLL/lymphoma11B (Bcl11b) participates in dental epithelial cell proliferation and differentiation. However, whether it affects the postnatal regulation of enamel matrix protein expression and ameloblast differentiation remains unclear. To clarify the role of Bcl11b in enamel development, the present study initially detected the protein expression levels of Bcl11b during tooth development using immunohistochemistry, from the embryonic lamina stage to the postnatal period, and demonstrated that Bcl11b is predominantly restricted to cervical loop epithelial cells at the cap and bell stages, whereas expression is reduced in ameloblasts. Notably, the expression pattern of Bcl11b during tooth development differed between rats and mice. Knockdown of Bcl11b by specific small interfering RNA attenuated the expression of enamel‑associated genes, including amelogenin, X‑linked (Amelx), ameloblastin (Ambn), enamelin (Enam), kallikrein related peptidase4 (Klk4), matrix metallopeptidase20 and Msh homeobox2 (Msx2). Chromatin immunoprecipitation assay verified that Msx2 was a transcriptional target of Bcl11b. However, overexpression of Msx2 resulted in downregulation of enamel‑associated genes, including Ambn, Amelx, Enam and Klk4. The present study suggested that Bcl11b serves a potentially important role in the regulation of ameloblast differentiation and enamel matrix protein expression. In addition, a complex feedback regulatory network may exist between Bcl11b and Msx2.

The biological significance of methylome differences in human papilloma virus associated head and neck cancer.

In recent years, studies have suggested that promoter methylation in human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) has a mechanistic role and has the potential to improve patient survival. The present study aimed to replicate key molecular findings from previous analyses of the methylomes of HPV positive and HPV negative HNSCC in an independent cohort, to assess the reliability of differentially methylated markers in HPV-associated tumors. HPV was measured using real-time quantitative PCR and the biological significance of methylation differences was assessed by Ingenuity Pathway Analysis (IPA). Using an identical experimental design of a 450K methylation platform, 7 of the 11 genes were detected to be significantly differentially methylated and all 11 genes were either hypo- or hypermethylated, which was in agreement with the results of a previous study. IPA's enriched networks analysis identified one network with msh homeobox 2 (MSX2) as a central node. Locally dense interactions between genes in networks tend to reflect significant biology; therefore MSX2 was selected as an important gene. Sequestration in the top four canonical pathways was noted for 5-hydroxytryptamine receptor 1E (serotonin signaling), collapsin response mediator protein 1 (semaphorin signaling) and paired like homeodomain 2 (bone morphogenic protein and transforming growth factor-β signaling). Placement of 9 of the 11 genes in highly ranked pathways and bionetworks identified key biological processes to further emphasize differences between HNSCC HPV positive and negative pathogenesis.

Mutation analysis by direct and whole exome sequencing in familial and sporadic tooth agenesis.

Dental agenesis is one of the most common congenital craniofacial abnormalities. Dental agenesis can be classified, relative to the number of missing teeth (excluding third molars), as hypodontia (1 to 5 missing teeth), oligodontia (6 or more missing teeth), or anodontia (lack of all teeth). Tooth agenesis may occur either in association with genetic syndromes, based on the presence of other inherited abnormalities, or as a non-syndromic trait, with both familiar and sporadic cases reported. In this study, we enrolled 16 individuals affected by tooth agenesis, prevalently hypodontia, and we carried out direct Sanger sequencing of paired box 9 (PAX9) and Msh homeobox 1 (MSX1) genes in 9 subjects. Since no mutations were identified, we performed whole exome sequencing (WES) in the members of 5 families to identify causative gene mutations either novel or previously described. Three individuals carried a known homozygous disease mutation in the Wnt family member 10A (WNT10A) gene (rs121908120). Interestingly, two of these individuals were siblings and also carried a heterozygous functional variant in EDAR-associated death domain (EDARADD) (rs114632254), another disease causing gene, generating a combination of genetic variants never described until now. The analysis of exome sequencing data in the members of other 3 families highlighted new candidate genes potentially involved in tooth agenesis and considered suitable for future studies. Overall, our study confirmed the major role played by WNT10A in tooth agenesis and the genetic heterogeneity of this disease. Moreover, as more genes are shown to be involved in tooth agenesis, WES analysis may be an effective approach to search for genetic variants in familiar or sporadic tooth agenesis, at least in more severe clinical manifestations.

Mutations in MSX1, PAX9 and MMP20 genes in Saudi Arabian patients with tooth agenesis

Tooth agenesis in human being is the most common congenital anomaly associated with dental development. Mutations in many genes such as MSH homeobox 1 (MSX1), paired box gene 9 (PAX9), ectodysplasin A (EDA) and EDA receptor (EDAR) have been associated with familial form of this condition. However, in large majority of patients, genetic cause could not be identified. The primary aim of present study was to identify the causative mutation(s) in these genes in Saudi Arabian families diagnosed with non-syndromic form of disease. Direct sequencing of coding regions, including exon-intron boundaries of these genes was carried out. All identified nucleotide variations were also tested to exclude possibility of being rare polymorphisms. The sequence analysis of exons and exon-intronic regions of these genes revealed five new mutations that include four in MSX1, one in PAX9 and one single nucleotide polymorphism (SNP) in majority of the patients in MMP20. One novel mutation in exon 1 of MSX1 gene (5354C > G; A40G) was found in three patients. In addition, another novel mutation was detected in two patients in exon 3 (PAX9) as g.10672A > T which changes asparagine to isoleucine at position 40. These mutations were not found in any of the control subjects. A single SNP in MMP20 genes (g.5066A > C) that changes lysine to threonine at position 18 was found in 10% controls as well. Our results for the first time demonstrates that mutations in MSX1 gene might play an important role in hypodontia cases involving pre-molars and is a risk factor for this ethnic population mainly of Arabs and is first report linking these mutations with tooth agenesis.

Overexpression of Dlx2leads to postnatal condyle degradation.

Distal-less homeobox 2 (Dlx2), a member of the Dlx family of transcription factors, is important for the development of craniofacial tissues. Previous studies based on knock-out mutant mice revealed that Dlx2 primarily disturbed the development of tissues from maxillary arch. The present study used a transgenic mouse model to specifically overexpress Dlx2 in neural crest cells in order to investigate the role of Dlx2 overexpression in post-natal condyle in mice. The model was constructed and the phenotype observed using gross observation, micro-CT scan and histological examination. The model determined that overexpression of Dlx2 may lead to postnatal condyle malformation, subchondral bone degradation and irregular histological structure of the condylar cartilage. In addition, the expression of osteocalcin in the condyle region was markedly downregulated, whereas expression of msh homeobox 2 was upregulated. The results of the present study suggest that Dlx2 overexpression in cranial neural crest cells would disrupt the development of post-natal condyle, which demonstrates that the expression level and the spatiotemporal expression patterns of Dlx2 may be important in regulating the development of post-natal condyle in mice, and also offered a possible temporal-mandibular joint osteoarthritis model animal for future studies.

Epigenetic Regulation of the Homeobox GeneMSX1Associates with Platinum-Resistant Disease in High-Grade Serous Epithelial Ovarian Cancer

Although high-grade serous ovarian cancer (HGSOC) is frequently chemoresponsive, a proportion of patients do not respond to platinum-based chemotherapy at presentation or have progression-free survival (PFS) of less than 6 months. Validated predictive biomarkers of lack of response would enable alternative treatment stratification for these patients and identify novel mechanisms of intrinsic resistance. Our aim was to identify DNA methylation biomarkers of poor response to chemotherapy and demonstrate involvement of the associated gene in platinum drug cell sensitivity. DNA methylation was investigated in independent tumor cohorts using Illumina HumanMethylation arrays and gene expression by Affymetrix arrays and qRT-PCR. The role of Msh homeobox 1 (MSX1) in drug sensitivity was investigated by gene reintroduction and siRNA knockdown of ovarian cancer cell lines. CpG sites at contiguous genomic locations within the MSX1 gene have significantly lower levels of methylation in independent cohorts of HGSOC patients, which recur by 6 months compared with after 12 months (P < 0.05, q < 0.05, n = 78), have poor RECIST response (P < 0.05, q < 0.05, n = 61), and are associated with PFS in an independent cohort (n = 146). A decrease in methylation at these CpG sites correlates with decreased MSX1 gene expression. MSX1 expression is associated with PFS (HR, 0.92; 95% CI, 0.85-0.99; P = 0.029; n = 309). Cisplatin-resistant ovarian cancer cell lines have reduced MSX1 expression, and MSX1 overexpression leads to cisplatin sensitization, increased apoptosis, and increased cisplatin-induced p21 expression. Hypomethylation of CpG sites within the MSX1 gene is associated with resistant HGSOC disease at presentation and identifies expression of MSX1 as conferring platinum drug sensitivity. Clin Cancer Res; 22(12); 3097-104. ©2016 AACR.

Functional analysis of a novel missense mutation in AXIN2 associated with non-syndromic tooth agenesis.

Tooth agenesis is a congenital anomaly frequently seen in humans. Several genes have been associated with non-syndromic tooth agenesis, including msh homeobox 1 (MSX1), paired box 9 (PAX9), axis inhibition protein 2 (AXIN2), ectodysplasin A (EDA), and wingless-type MMTV integration site family member 10A (WNT10A). In this study, we investigated a Chinese family with non-syndromic tooth agenesis. A novel missense mutation (c.C1978T) in AXIN2 was identified in affected members. The mutation results in a His660Tyr substitution located between the Axin beta-catenin binding domain and the DIX domain of the axis inhibition protein 2 (AXIN2). We analysed this novel AXIN2 mutant, together with two reported AXIN2 mutants [c.1966C>T (p.Arg656Stop) and c.1994delG (p.Leu688Stop)] that cause colorectal cancer with and without oligodontia, to study the effect of the mutant p.His660Tyr on the Wnt/β-catenin signaling pathway and to compare the molecular pathogenesis of different AXIN2 mutants in tooth agenesis and carcinogenesis. Further in vitro experiments indicated that the mutant p.His660Tyr caused inhibition of the Wnt/β-catenin pathway, and the mutants p.Arg656Stop and p.Leu688Stop resulted in over-activation of the Wnt/β-catenin pathway. In line with previous AXIN2 mutation studies, we suggest that AXIN2 mutations with different levels of severity may have distinct effects on the Wnt pathway and the phenotype of disease. Our study provides functional evidence supporting the notion that both inhibition and over-activation of the Wnt pathway may lead to tooth agenesis.

MSX2 Induces Trophoblast Invasion in Human Placenta.

Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2

Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Beta-glycerophosphate (β-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle α-actin (SM-α-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by β-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification.

Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells.

Msh homeobox 1 (MSX1) encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL), and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4–12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells.

GENETIC DISORDERS OF PITUITARY DEVELOPMENT IN PATIENTS WITH SHEEHAN'S SYNDROME.

Genetic disorders associated with the development of the pituitary gland and cranial bones may cause a genetic tendency toward Sheehan's syndrome (SS). Our aim in this study was to investigate expression disorders in the genes responsible for the development of the pituitary gland and cranial bones in patients with SS. Forty-four patients who were previously diagnosed with SS and 43 healthy women were compared in terms of the mean expression values of genes including the prophet of PIT-1 (PROP1), HESX homeobox 1 (HESX1), POU class 1 homeobox 1 (POU1F1), LIM homeobox 3 (LHX3), LHX4, glioma-associated oncogene homolog 2 (GLI2), orthodenticle homeobox 2 (OTX2), SIX homeobox 3 (SIX3), SIX6, T-box transcription factor 19 (TBX19), transducin-like enhancer protein 1 (TLE1), TLE3, distal-less homeobox 2 (DLX2), DLX5, MSH homeobox 2 (MSX2), and paired box 3 (PAX3). The mean expression values of the HESX1, TLE1, TLE3, and MSX2 genes were significantly different in the SS group from the healthy control group, while the mean expression values of the remaining genes were similar. The present study concludes that abnormal expressions of HESX1, TLE1, TLE3, and MSX2 genes may cause a genetic predisposition to the development of SS.

Msx1 homeodomain transcription factor and TATA-binding protein interact to repress the expression of the glycoprotein hormone α subunit gene

Studying the regulatory mechanism of the glycoprotein hormone α subunit (αGSU) gene in thyrotropes is essential for understanding the synthesis of functional thyroid-stimulating hormone (TSH). Here, we investigated the influence of a homeodomain transcription factor Msx1 (Msh homeobox 1) on αGSU expression in thyrotropes. The transient expression of Msx1 inhibited the activity of an αGSU reporter gene, as well as its endogenous mRNA level in thyrotrope-derived αTSH cells. Luciferase reporter assays with serial deletion constructs and a close examination of the sequences revealed that the putative Msx1 binding site (PMS) in the αGSU promoter is not responsible for Msx1-mediated transcriptional repression. We also identified the TATA-box binding protein (TBP) as an interacting protein in thyrotropes. Interaction of TBP with Msx1 attenuates the inhibitory effect of Msx1 on αGSU gene expression in a DNA binding-independent manner. Furthermore, transient transfection studies with mutant Msx1 revealed that the interaction of TBP and Msx1 is critical for Msx1-mediated transcriptional repression of the αGSU. These results suggest that Msx1 functions as a transcriptional repressor of αGSU and that its interaction with TBP is an integral part of the mechanism by which Msx1 regulates the inhibition of αGSU gene expression.

Abstract A13: MSX1-induced neural crest-like reprograming promotes melanoma progression

Abstract Melanoma cells share many biological properties with neural crest cells. Here, we show that the homeodomain transcription factor Msh homeobox 1 (MSX1), which is essential for neural crest specification, reprograms melanocytes towards a neural crest precursor-like state. MSX1-reprogrammed melanocytes express the neural crest marker p75 and are able to differentiate into neuronal and mesenchymal lineages. Mechanistically, MSX1 suppresses the proximal promoter of microphthalmia-associated transcription factor (MITF), the master transcriptional regulator of melanogenesis. MSX1 expression is also significantly correlated with melanoma progression. MSX1 prompts melanoma cell motility and depletion of MSX1 significantly inhibits melanoma metastasis. These results demonstrate that not only can neural crest-like reprogramming in melanocytes be achieved by a single factor, but also that similar dedifferentiation is a critical process for melanoma progression. Citation Format: Markus V. Heppt, Wang X. Joshua, Denitsa M. Hristova, Zhi Wei, Martin Irmler, Carola Berking, Robert Besch, Johannes Beckers, Frank J. Rauscher, III, David E. Fisher, Meenhard Herlyn, Mizuho Fukunaga-Kalabis. MSX1-induced neural crest-like reprograming promotes melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A13.

Identifying novel biomarkers of gastric cancer through integration analysis of single nucleotide polymorphisms and gene expression profile.

Single nucleotide polymorphisms (SNPs) are an important cause of functional variation in proteins leading to tumorigenesis. We aimed to identify candidate biomarkers with polymorphisms in gastric cancer (GC). The SNP microarray profile GSE29996 including 50 GC samples and 50 normal controls, and gene expression data GSE56807 consisting of 5 GC samples and 5 controls were downloaded from the Gene Expression Omnibus database. After preprocessing of raw data, GC-associated SNPs were identified using the Cochran-Armitage trend test, and differentially expressed genes (DEGs) were screened out using the limma package in R. Significant DEGs with risk associated SNP loci were screened using the Fisher combination test. Gene ontology function and pathway enrichment analyses were performed for DEGs with risk associated SNP loci by GenCLip online tool. Transcriptional regulatory analysis was also conducted for transcription factor and target DEGs. A total of 79 DEGs with risk associated SNP loci were identified from GC samples compared with normal controls. These DEGs were mainly enriched in anatomical structure development, including embryo development. Additionally, DEGs were significantly involved in the NO1 pathway, including actin, alpha 1, skeletal muscle (ACTA1). In the regulatory network, transcription factor forkhead box L1 (FOXL1) regulated 26 DEGs with risk associated SNP loci, including Iroquois homeobox 1 (IRX1) rs11134044, sex determining region Y (SRY)-box1 (SOX1) rs9549447 and msh homeobox 1 (MSX1) rs41451149. IRX1, SOX1 and MSX1 with risk associated SNP loci may serve as candidate biomarkers for diagnosis and prognosis of GC.