Paired Box Homeotic Gene Research Articles

HC-HA/PTX3 from amniotic membrane reverts senescent limbal niche cells to Pax6+ neural crest progenitors to support limbal epithelial progenitors

Quiescence and self‐renewal of human corneal epithelial progenitor/stem cells (LEPC) are regulated by the limbal niche, presumably through close interaction with limbal (stromal) niche cells (LNC). Paired box homeotic gene 6 (Pax6), a conserved transcription factor essential for eye development, is essential for proper differentiation of limbal and corneal epithelial stem cells. Pax6 haploinsufficiency causes limbal stem cell deficiency, which leads to subsequent corneal blindness. We previously reported that serial passage of nuclear Pax6+ LNC resulted in the gradual loss of nuclear Pax6+ and neural crest progenitor status, the latter of which was reverted upon recovery of Pax6. These findings suggest Pax6 plays a pivotal role in supporting the self‐renewal of LEPC in limbal niche. Herein, we show that HC‐HA/PTX3, a unique matrix purified from amniotic membrane (AM) and consists of heavy chain 1of inter‐α‐trypsin inhibitor covalently linked to hyaluronic acid and complexed with pentraxin 3, is capable of reverting senescent LNC to nuclear Pax6+ neural crest progenitors that support self‐renewal of LEPC. Such reversion is causally linked to early cell aggregation mediated by activation of C‐X‐C chemokine receptor type 4 (CXCR4)‐mediated signaling followed by activation of bone morphogenetic protein (BMP) signaling. Furthermore, CXCR4‐mediated signaling, but not BMP signaling, controls recovery of the nuclear Pax6+ neural crest progenitors. These findings not only explain why AM helps in vivo and ex vivo expansion of human LEPC, but they also illuminate the potential role of HC‐HA/PTX3 as a surrogate matrix niche that complements stem cell‐based therapies in regenerative medicine.

Independent association of whole blood miR-328 expression and polymorphism at 3′UTR of the PAX6 gene with myopia

PurposeThis study aimed to find associations between miR-328 expression in whole blood, polymorphism at 3′UTR of the PAX6 gene (paired box homeotic gene 6) and myopia. MethodsWe evaluated 451 individuals (142 individuals with low, 49 with moderate and 13 with high-degree myopia, and 247 healthy individuals). DNA and RNA were extracted from peripheral blood samples. Expression of miR-328 was assessed and genotyping of single-nucleotide polymorphisms (SNPs) of the PAX6 (rs662702) performed using the Applied Biosystems 7900HT Real-Time Polymerase Chain Reaction System. ResultsModerate and high degree myopia showed significant differences between TT and CT genotypes of the PAX6 gene (p < 0.001). In the myopia group, 71.4% of the subjects had the TT genotype and 28.6% had the CT genotype; meanwhile in the control group, 97.1% had the TT genotype and 2.9% had the CT genotype. The odds ratio of having moderate and/or high degree myopia for individuals with the CT genotype was 13.6 (2.865–64.55) 95% CI versus TT genotype (p = 0.001). MiR-328 results showed that ∆Ct values differed statistically significantly between the myopia and control groups. Patients with myopia in the peripheral blood cells had a higher expression of miR-328 than controls (p < 0.05). ConclusionsSignificant differences were detected between the PAX6 gene (rs662702) TT and CT genotypes in moderate and high degree myopia; the risk C allele increased the risk for myopia. The expression level of miR-328 in peripheral blood cells was higher in patients with myopia than controls. We did not find the association between expression of mir-328 in the peripheral blood cells and PAX6 gene (rs662702) polymorphism comparing myopia and control groups.

Genetic Differentiation of Common Fox Vulpes Vulpes (Linnaeus, 1758) on the Basis of the Insulin-Like Growth Factor 1 (Igf1), Myosin-Xv (Myo15a) and Paired Box Homeotic 3 (Pax3) Genes Fragments Polymorphism

Abstract Single-nucleotide polymorphism (SNP) was analysed for selected fragments of three genes - insulin-like growth factor 1 (IGF1), myosin-XV (MYO15A) and paired box homeotic gene 3 (PAX3) - in farm and wild red foxes from two continents. The study was undertaken in order to verify whether the SNP characteristics of these genes enable farm-bred foxes to be distinguished from free-living foxes. The greatest number of changes were detected in the IGF1 gene. For each of the genes investigated specific SNP profiles characteristic only for farm foxes and only for wild foxes were noted. At the same time, specific SNP profiles were noted for wild foxes from North America and from Europe. The frequency of SNP (bases per SNP) in the gene fragments examined was 22 bp for IGF1, 34 bp for PAX3 and 56 bp for MYO15A. Single-nucleotide polymorphism is a very good molecular marker enabling characterization of nucleotide variation in the genes investigated between wild and farm individuals

Stat3-Targeted Therapies Overcome the Acquired Resistance to Vemurafenib in Melanomas

Vemurafenib (PLX4032), a selective inhibitor of Braf, has been approved by the US Food and Drug Administration for the treatment of unresectable or metastatic melanoma in patients with Braf(V600E) mutations. Many patients treated with vemurafenib initially display dramatic improvement, with decreases in both risk of death and tumor progression. Acquired resistance, however, rapidly arises in previously sensitive cells. We attempted to overcome this resistance by targeting the signal transducer and activator of transcription 3 (STAT3)-paired box homeotic gene 3 (PAX3)-signaling pathway, which is upregulated, owing to fibroblast growth factor 2 (FGF2) secretion or increased kinase activity, with the Braf(V600E) mutation. We found that activation of Stat3 or overexpression of PAX3 induced resistance to vemurafenib in melanoma cells. In addition, PAX3 or Stat3 silencing inhibited the growth of melanoma cells with acquired resistance to vemurafenib. Furthermore, treatment with the Stat3 inhibitor, WP1066, resulted in growth inhibition in both vemurafenib-sensitive and -resistant melanoma cells. Significantly, vemurafenib stimulation induced FGF2 secretion from keratinocytes and fibroblasts, which might uncover, at least in part, the mechanisms underlying targeting Stat3-PAX3 signaling to overcome the acquired resistance to vemurafenib. Our results suggest that Stat3-targeted therapy is a new therapeutic strategy to overcome the acquired resistance to vemurafenib in the treatment of melanoma.

TBX2 expression is regulated by PAX3 in the melanocyte lineage.

The paired box homeotic gene 3 (PAX3) is a crucial regulator for the maintenance of melanocytic progenitor cells and has a poorly defined role in melanoma. To understand how PAX3 affects melanocyte and melanoma proliferation, we identified potential PAX3 downstream targets through gene expression profiling. Here, we identify T-box 2 (TBX2), a key developmental regulator of cell identity and an antisenescence factor in melanoma, as a directly regulated PAX3 target. We also found that TBX2 is involved in the survival of melanoma cells and is overexpressed in some melanoma specimens. The identification of TBX2 as a target for PAX3 provides a key insight into how PAX3 may contribute to melanoma evolution and may provide opportunities for prosenescence therapeutic intervention aimed at disrupting the ability of PAX3 to regulate TBX2.

Two novel mutations of the PAX6 gene causing different phenotype in a cohort of Chinese patients

Aniridia (AN) is a rare congenital panocular disorder caused by the mutations of the paired box homeotic gene 6(PAX6) gene. The PAX6gene is also involved in other anterior segment malformations including Peters anomaly. We studied the PAX6gene mutations in a cohort of affected individuals with different clinical phenotype including AN, coloboma of iris and choroid, or anterior segment malformations. Six unrelated families and 10 sporadic patients were examined clinically. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Mutation screening of all exons of the PAX6gene was performed by direct sequencing of PCR-amplified DNA fragments. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large deletions. By clinical examination, the patients and the pedigrees were divided into the following three groups: AN, coloboma of iris and choroids, and the anterior segment malformations including peters anomaly. Sequencing of the PAX6gene, three intragenic mutations including a novel heterozygous splicing-site mutations c.357-3C>G (p.Ser119fsX) were identified in the patients of the AN group. A novel missense mutation c.643T>C (p.S216P) was detected in the anterior segment malformation group. The mutation p.S216P located in the homeodomain region of the PAX6 caused the phenotype of Peters anomaly in family A6 with different expressing. Through MLPA analysis, a large deletion including the whole PAX6gene and DKFZ p686k1684gene was detected in one sporadic patient from the AN group. Neither intragenic mutation nor large deletion was identified in the group with coloboma of iris and choroid. Our findings further confirmed that different kind of mutations might cause different ocular phenotype, and clearly clinical phenotype classification might increase the mutation detection rate of the PAX6gene.

Inhibition of PAX3 by TGF-β Modulates Melanocyte Viability

The protein encoded by paired-box homeotic gene 3 (PAX3) is a key regulator of the microphthalmia-associated transcription factor (Mitf) in the melanocyte lineage. Here, we show that PAX3 expression in skin is directly inhibited by TGF-beta/Smads. UV irradiation represses TGF-beta in keratinocytes, and the repression of TGF-beta/Smads upregulates PAX3 in melanocytes, which is associated with a UV-induced melanogenic response and consequent pigmentation. Furthermore, the TGF-beta-PAX3 signaling pathway interacts with the p53-POMC/MSH-MC1R signaling pathway, and both are crucial in melanogenesis. The activation of p53-POMC/MSH-MC1R signaling is required for the UV-induced melanogenic response because PAX3 functions in synergy with SOX10 in a cAMP-response element (CRE)-dependent manner to regulate the transcription of Mitf. This study will provide a rich foundation for further research on skin cancer prevention by enabling us to identify targeted small molecules in the signaling pathways of the UV-induced melanogenic response that are highly likely to induce naturally protective pigmentation.

Estrogen influences satellite cell activation and proliferation following downhill running in rats

To investigate the influence of estrogen on postexercise muscle repair processes, we examined the effects of estrogen supplementation (0.25-mg pellet) on numbers of myofibers positive for markers of total, activated, and proliferating satellite cells in rat skeletal muscles 72 h following downhill running. Ovariectomized female rats (n = 44) were divided into four groups (n = 11 per group): sham (no estrogen) controls (SC); sham, exercised (SE); estrogen-supplemented controls (EC); and estrogen-supplemented, exercised (EE). After 8 days of estrogen exposure, animals were exposed to 90 min of treadmill running at 17 m/min (-13.5 degrees ). Seventy-two hours later, soleus and white vastus muscles were removed and immunostained for total [paired box homeotic gene 7 (Pax7)], [activated myogenic differentiation factor D (MyoD)], and proliferating [5-bromo-2'-deoxyuridine (BrdU)] satellite cells. beta-Glucuronidase activity was increased (P < 0.05) in both muscles following exercise; however, the postexercise elevations in enzyme activity were attenuated in the EE group compared with the SE group in the soleus (P < 0.05). Immunohistochemical analysis revealed that exercised groups displayed increased numbers of myofibers containing total, activated, and proliferating satellite cells compared with control groups (P < 0.05). Furthermore, greater numbers of fibers positive for markers of total, activated, and proliferating satellite cells were observed postexercise in EE animals compared with SE animals for both muscles (P < 0.05). The results demonstrate that estrogen may potentially influence post-damage repair of skeletal muscle through activation of satellite cells.

Serial Monitoring of Circulating Melanoma Cells During Neoadjuvant Biochemotherapy for Stage III Melanoma: Outcome Prediction in a Multicenter Trial

Circulating tumor cells (CTCs) in blood may be important in assessing tumor progression and treatment response. We hypothesized that quantitative real-time reverse transcriptase polymerase chain reaction using multimarker mRNA assays could detect CTCs and be used as a surrogate predictor of outcome in patients receiving neoadjuvant biochemotherapy (BC) for melanoma. Blood specimens were collected at four sampling points from 63 patients enrolled on a prospective multicenter phase II trial of BC before and after surgical treatment of American Joint Committee on Cancer stage III melanoma. Each specimen was assessed by quantitative real-time reverse transcriptase polymerase chain reaction for expression of four melanoma-associated markers: melanoma antigen recognized by T cells 1; beta1 --> 4-N-acetylgalactosaminyltransferase; paired box homeotic gene transcription factor 3; and melanoma antigen gene-A3 family, and the changes of CTCs during treatment and prognostic effect of CTCs after overall treatment on recurrence and survival were investigated. At a median postoperative follow-up time of 30.4 months, 44 (70%) patients were clinically disease free. In relapse-free patients, the number of detected markers significantly decreased during preoperative BC (P = .036), during postoperative BC (P = .002), and during overall treatment (P < .0001). Marker detection after overall treatment was associated with significant decreases in relapse-free and overall survival (P < .0001). By multivariate analysis using a Cox proportional-hazards model, the number of markers detected after overall treatment was a significant independent prognostic factor for overall survival (risk ratio, 12.6; 95% CI, 3.16 to 50.5; P = .0003). Serial monitoring of CTCs in blood may be useful for indicating systemic subclinical disease and predicting outcome of patients receiving neoadjuvant BC for metastatic melanoma.

Differentiation of Human Liver-Derived, Insulin-Producing Cells Toward the β-Cell Phenotype

beta-Cell transplantation is viewed as a cure for type 1 diabetes; however, it is limited by the number of pancreas donors. Human stem cells offer the promise of an abundant source of insulin-producing cells, given the existence of methods for manipulating their differentiation. We have previously demonstrated that the expression of the beta-cell transcription factor pancreatic duodenal homeobox 1 (PDX-1) in human fetal liver cells activates multiple aspects of the beta-cell phenotype. These cells, termed FH-B-TPN cells, produce insulin, release insulin in response to physiological glucose levels, and replace beta-cell function in diabetic immunodeficient mice. However, they deviate from the normal beta-cell phenotype by the lack of expression of a number of beta-cell genes, the expression of non-beta-cell genes, and a lower insulin content. Here we aimed to promote differentiation of FH-B-TPN cells toward the beta-cell phenotype using soluble factors. Cells cultured with activin A in serum-free medium upregulated expression of NeuroD and Nkx2.2 and downregulated paired box homeotic gene 6 (PAX-6). Glucokinase and prohormone convertase 1/3 were also upregulated, whereas pancreatic polypeptide and glucagon as well as liver markers were downregulated. Insulin content was increased by up to 33-fold, to approximately 60% of the insulin content of normal beta-cells. The cells were shown to contain human C-peptide and release insulin in response to physiological glucose levels. Cell transplantation into immunodeficient diabetic mice resulted in the restoration of stable euglycemia. The cells continued to express insulin in vivo, and no cell replication was detected. Thus, the manipulation of culture conditions induced a significant and stable differentiation of FH-B-TPN cells toward the beta-cell phenotype, making them excellent candidates for beta-cell replacement in type 1 diabetes.

Satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice

To determine whether muscle satellite cells and utrophin are correlated with the degree of damage in mdx skeletal muscles, we measured the area of the degenerative region as an indicator of myofiber degeneration in the masseter, gastrocnemius, soleus, and diaphragm muscles of mdx mice. Furthermore, we analyzed the expression levels of the paired box homeotic gene 7 (pax7), m-cadherin (the makers of muscle satellite cells), and utrophin mRNA. We also investigated the immunolocalization of m-cadherin and utrophin proteins in the muscles of normal C57BL/10J (B10) and mdx mice. The expression level of pax7 mRNA and the percentage of m-cadherin-positive cells among the total number of cell nuclei in the muscle tissues in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there was no significant correlation between muscle damage and expression level for pax7 mRNA (R = -0.140), nor was there a correlation between muscle damage and the percentage of satellite cells among the total number of cell nuclei (R = -0.411) in the mdx mice. The expression level of utrophin mRNA and the intensity of immunostaining for utrophin in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there also was not a significant correlation between muscle damage and expression level of utrophin mRNA (R = 0.231) in the mdx mice, although upregulated utrophin was incorporated into the sarcolemma. These results suggest that satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice.

Analysis of the 5′ region of the canine PAX3 gene and exclusion as a candidate for Dalmatian deafness1

The causative mutation in a gene related to hearing loss in Dalmatians has been elusive. Because of its role in melanocyte migration and differentiation as integral component of the inner ear, we hypothesized that the canine PAX3 (paired box homeotic gene 3) gene could be a candidate for Dalmatian deafness. Therefore, we isolated the canine PAX3 gene and searched for causative mutations within the coding region of important regulatory domains of PAX3. However, no mutations were identified when comparing the DNA sequences of healthy and affected dogs. These results were confirmed by a two-point linkage analysis in 203 Dalmatians transmitting deafness. Our data clearly show that the canine PAX3 gene can be excluded as candidate for Dalmatian deafness.

Cloning of the cDNA encoding phenylalanyl tRNA synthetase regulatory alpha-subunit-like protein whose expression is down-regulated during differentiation.

Hybrid polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA), induce differentiation of transformed cells. Differential display of RNA was used to identify genes whose expression is changed during SAHA-induced differentiation of murine erythroleukemia (MEL) cells. One such cDNA was identified whose mRNA level decreased by 50% after 8h of SAHA treatment as determined by Northern blot analysis. The full-length cDNA (1944bp in length) was cloned by sequencing of an EST clone and rapid amplification of 5' cDNA ends (5'-RACE). The predicted amino acid sequence is 589 amino acids and shares 45% identity with the yeast cytoplasmic phenylalanyl tRNA synthetase (PheRS) regulatory alpha-subunit. Human EST clones which share over 90% identity of predicted amino acid sequence with this cDNA map to chromosome 2 near the paired box homeotic gene 3 (PAX3) locus, a region syngenic to mouse chromosome 1. This is the first report of the cloning of the full-length cDNA for the murine PheRS regulatory alpha-subunit-like protein. The level of PheRS alpha-subunit-like mRNA is regulated during differentiation but not during cell cycle progression.

Analysis of select folate pathway genes, PAX3, and human T in a midwestern neural tube defect population

Neural tube defects (NTDs) are a common birth defect, seen in approximately 1/1,000 births in the United States. NTDs are considered a complex trait where several genes, interacting with environmental factors, create the phenotype. Using a Midwestern NTD population consisting of probands, parents, and siblings from Iowa, Minnesota, and Nebraska, we analyzed a range of candidate genes, including 5,10–methylenetetrahydrofolate reductase (MTHFR), folate receptors-α (FOLR1; hereafter abbreviated “FR-α”) and -β (FOLR2; hereafter, “FR-β”), methionine synthase (hereinafter, “MS”), T, the human homolog of the murine Brachyury gene, and the paired-box homeotic gene 3 (PAX3), for association with NTDs. We were unable to demonstrate an association using a previously described Ala→Val mutation in MTHFR and the majority of our NTD populations. However, we discovered a silent polymorphism in exon 6 of MTHFR which conserved a serine residue and which showed significant association with NTDs in our Iowa population. Analysis of exon 7 of MTHFR then demonstrated an Ala→Glu mutation which was signficantly associated with our Iowa NTD population; however, we could not replicate this result either in a combined Minnesota/Nebraska or in a California NTD population. Using polymorphic markers for MS, FR-β, T, and PAX3, we were unable to demonstrate linkage disequilibrium with our NTD populations. A mutation search of FR-α revealed one proband with a de novo silent mutation of the stop codon. This work provides a new panel of genetic variants for studies of folate metabolism and supports, in some NTD populations, an association between MTHFR and NTDs. Teratology 59:331–341, 1999. © 1999 Wiley-Liss, Inc.

Analysis of select folate pathway genes, PAX3, and human T in a Midwestern neural tube defect population.

Neural tube defects (NTDs) are a common birth defect, seen in approximately 1/1,000 births in the United States. NTDs are considered a complex trait where several genes, interacting with environmental factors, create the phenotype. Using a Midwestern NTD population consisting of probands, parents, and siblings from Iowa, Minnesota, and Nebraska, we analyzed a range of candidate genes, including 5,10-methylenetetrahydrofolate reductase (MTHFR), folate receptors-alpha (FOLR1; hereafter abbreviated "FR-alpha") and -beta (FOLR2; hereafter, "FR-beta"), methionine synthase (hereinafter, "MS"), T, the human homolog of the murine Brachyury gene, and the paired-box homeotic gene 3 (PAX3), for association with NTDs. We were unable to demonstrate an association using a previously described Ala-->Val mutation in MTHFR and the majority of our NTD populations. However, we discovered a silent polymorphism in exon 6 of MTHFR which conserved a serine residue and which showed significant association with NTDs in our Iowa population. Analysis of exon 7 of MTHFR then demonstrated an Ala-->Glu mutation which was significantly associated with our Iowa NTD population; however, we could not replicate this result either in a combined Minnesota/ Nebraska or in a California NTD population. Using polymorphic markers for MS, FR-beta, T, and PAX3, we were unable to demonstrate linkage disequilibrium with our NTD populations. A mutation search of FR-alpha revealed one proband with a de novo silent mutation of the stop codon. This work provides a new panel of genetic variants for studies of folate metabolism and supports, in some NTD populations, an association between MTHFR and NTDs.

Corneal Epithelium-specific Mouse Keratin K12 Promoter

Keratins are structural proteins expressed by epithelial cells. Approximately 30 different keratin proteins have been identified, each with a specific expression pattern in different epithelial cells. The tissue-specific promoter of several keratin genes have been used to direct the expression of transgenes in animals. Keratin K12 and K3 are expressed in differentiated and stratified corneal epithelium, although the relative expression of each appears to vary between species. We targeted the mouse K12 keratin gene in order to develop a tissue-specific promoter that could be utilized to study the functions of genes of interest expressed in the corneal epithelium. Three 5′ truncated fragments of the keratin K12 promoter (1.03, 0.71 and 0.25 Kb) showed higher functional and tissue-specific promoter activity in a human corneal epithelial cell line than other cell lines. The 0.25 Kb K12 promoter fragment was also active in cultured rabbit corneal epithelial cells. Thus, increased expression in corneal epithelial cells directed by fragments of the mouse K12 promoter extended across species lines. The paired box homeotic gene 6 (PAX-6), which is involved in controlling eye development, stimulated the activity of keratin K12 promoter.