HYAL1 Research Articles

DNA Methylation Status of HYAL1 in Malignant and Benign Thyroid Nodules.

Differentiation between benign and malignant thyroid nodules has been a challenge in clinical practice. Exploring a novel biomarker to determine the malignancy of thyroid nodules has important implications. We semi-quantitatively determined the DNA methylation levels of four CpG sites located at the gene body of HYAL1 in formalin-fixed paraffin-embedded (FFPE) tissue samples from 190 early-stage papillary thyroid cancer (PTC) cases and 190 age- and gender-matched subjects with benign thyroid nodule (BTN). HYAL1 expression was evaluated by immunohistochemical (IHC) staining in another cohort of 55 PTC and 55 matched BTN cases. Covariates-adjusted odds ratios (ORs) for 10% increased methylation were calculated by binary logistic regression. A 165 bp amplicon covering four CpG sites at the second exon of HYAL1 gene was designed. After adjusted for all covariates, higher methylation level of HYAL1_CpG_3,4 in the FFPE tissue was associated with PTC (OR per 10% increased methylation=1.53, p=0.025), even with stage І PTC (OR per 10% increased methylation=1.58, p=0.021). Hypermethylation of HYAL1_CpG_3,4 had a significant association with early-stage PTC in the females (OR per 10% increased methylation=1.60, p=0.028) rather than in the males. Besides, we found the higher expression of HYAL1 protein in PTC than that in BTN patients (IHC score: 2.3 vs. 0.5, p=1.00E-06). Our study suggested altered methylation and expression of HYAL1 could be a novel and potential biomarker in distinguishing malignant and benign thyroid nodules.

Hyaluronan in articular cartilage: Analysis of hip osteoarthritis and osteonecrosis of femoral head.

Hyaluronan (HA) plays crucial roles in the maintenance of high-quality cartilage extracellular matrix. Several studies have reported the HA in synovial fluid in patients with osteoarthritis (OA), but few have described the changes of HA in articular cartilage of OA or idiopathic osteonecrosis of the femoral head (ONFH). KIAA1199 was recently reported to have strong hyaluronidase activity. The aim of this study was to clarify the HA metabolism in OA and ONFH, particularly the involvement of KIAA1199. Immunohistochemical analysis of KIAA1199 and HA deposition was performed for human OA (n = 10), ONFH (n = 10), and control cartilage (n = 7). The concentration and molecular weight (MW) of HA were determined by competitive HA ELISA and Chromatography, respectively. Regarding HA metabolism-related molecules, HAS1, HAS2, HAS3, HYAL1, HYAL2, and KIAA1199 gene expression was assessed by reverse transcriptase polymerase chain reaction. Histological analysis showed the overexpression of KIAA1199 in OA cartilage, which was accompanied by decreased hyaluronic acid binding protein (HABP) staining compared with ONFH and control. Little KIAA1199 expression was observed in cartilage at the collapsed area of ONFH, which was accompanied by a slight decrease in HABPstaining. The messenger RNA (​​​​​mRNA) expression of HAS2 and KIAA1199 was upregulated in OA cartilage, while the mRNA expression of genes related to HA catabolism in ONFH cartilage showed mostly a downward trend. The MWof HA in OA cartilage increased while that in ONFH cartilage decreased. HA metabolism in ONFH is suggested to be generally indolent, and is activated in OA including high expression of KIAA1199. Interestingly, MW of HA in OA cartilage was not reduced.

Detection of DNA methylation of HYAL2 gene for differentiating malignant from benign thyroid tumors

To assess the value of DNA methylation level of HYAL2 gene as a molecular marker for differential diagnosis of malignant and benign thyroid tumors. DNA methylation of HYAL2 gene in tissue specimens of 190 patients with papillary thyroid cancer (PTC) and 190 age- and gender-matched patients with benign thyroid tumors was examined by mass spectrometry, and the protein expression of HYAL2 was detected immunohistochemically for another 55 pairs of patients. Logistic regression analysis was performed to calculate the odds ratio (OR) and evaluate the correlation of per 10% reduction in DNA methylation with PTC. Receiver operating characteristic (ROC) curve analysis was performed and the area under curve (AUC) was calculated to assess the predictive value of alterations in HYAL2 methylation. Hypomethylation of HYAL2_CpG_3 was significantly correlated with early-stage PTC (OR=1.51, P=0.001), even in stage I cancer (OR=1.42, P=0.007). Age-stratified analysis revealed a significantly stronger correlation between increased HYAL2_CpG_ 3 methylation and early-stage PTC in patients below 50 years than in those older than 50 years (OR: 1.89 vs 1.37, P < 0.05); ROC analysis also showed a larger AUC of 0.787 in younger patients. The results of immunohistochemistry showed that patients with PTC had significantly higher protein expressions of HYAL2 than patients with benign tumors. The alterations of DNA methylation level of HYAL2 gene is significantly correlated with early-stage PTC, suggesting the value of DNA methylation level as a potential biomarker for differentiation of malignant from benign thyroid tumors.

Expression and activity of hyaluronidases HYAL-1, HYAL-2 and HYAL-3 in the human intervertebral disc

PurposeHyaluronic acid plays an essential role in water retention of the intervertebral disc (IVD) and thus provides flexibility and shock absorbance in the spine. Hyaluronic acid gets degraded by hyaluronidases (HYALs), and some of the resulting fragments were previously shown to induce an inflammatory and catabolic response in human IVD cells. However, no data currently exist on the expression and activity of HYALs in IVD health and disease.MethodsGene expression, protein expression and activity of HYALs were determined in human IVD biopsies with different degrees of degeneration (n = 50 total). Furthermore, freshly isolated human IVD cells (n = 23 total) were stimulated with IL-1β, TNF-α or H2O2, followed by analysis of HYAL-1, HYAL-2 and HYAL-3 gene expression.ResultsGene expression of HYAL-1 and protein expression of HYAL-2 significantly increased in moderate/severe disc samples when compared to samples with no or low IVD degeneration. HYAL activity was not significantly increased due to high donor–donor variation, but seemed overall higher in the moderate/severe group. An inflammatory environment, as seen during IVD disease, did not affect HYAL-1, HYAL-2 or HYAL-3 expression, whereas exposure to oxidative stress (100 µM H2O2) upregulated HYAL-2 expression relative to untreated controls.ConclusionAlthough HYAL-1, HYAL-2 and HYAL-3 are all expressed in the IVD, HYAL-2 seems to have the highest pathophysiological relevance. Nonetheless, further studies will be needed to comprehensively elucidate its significance and to determine its potential as a therapeutic target.Graphic abstractThese slides can be retrieved under Electronic Supplementary Material.

Global gene expression profile in canine mammary carcinomas

Mammary gland tumors are a heterogeneous group of neoplastic diseases. Genetic studies make it possible to determine genetic profiles and identify new molecular markers. The aim of the study was to evaluate the gene expression profile of canine mammary carcinomas and identify potential prognostic markers. Twelve mammary cancer samples from bitches were collected for the evaluation of global gene expression. Microarray assays were performed using commercial kits. Statistical analysis of the microarray was done using moderate t-statistic and adjusted using the Benjamini and Hochberg procedure. Differential connectivity analysis was also performed. Enrichment analyses were conducted using WebGestalt. P-values were calculated using hypergeometric statistics and adjusted using the Benjamini and Hochberg procedure. The HYAL-1 gene was validated using quantitative PCR (qPCR).There were 878 upregulated genes and 821 downregulated genes in the neoplasms studied. Enrichment analysis (individual analysis) identified the HYAL-1 gene as a potential marker of tumorigenesis and tumor recurrence. Differential connectivity analysis demonstrated 262 differentially connected genes.

Brak związku pomiędzy pochodzącą od człowieka neandertalskiego transwersją g.50318299T&gt;G genu HYAL2 a predyspozycją do bliznowca

Wstęp: Pochodzący od Homo neanderthalensis DNA stanowi ok. 1–4% genomu w populacjach euroazjatyckich. Markerem rozpowszechnionego u mieszkańców Azji Wschodniej neandertalskiego introgresywnego haplotypu na chromosomie 3p21.31 jest transwersja g.50318299T&gt;G (c.1252A&gt;C, rs35455589) genu HYAL2 kodującego hialuronidazę typu 2. Transwersja HYAL2 jest ściśle sprzężona z polimorfizmem rs11130248 (g.50314769A&gt;G) predysponującym do bliznowca w populacji Japończyków. Stąd też rodzą się pytania, czy transwersja g.50318299T&gt;G HYAL2 występuje również u współczesnych Polaków i czy predysponuje do bliznowca w naszej populacji.Materiały i metody: Identyfikację transwersji g.50318299T&gt;G HYAL2 przeprowadzono metodą sekwencjonowania w próbkach genomowego DNA wyizolowanego z krwi pępowinowej 200 zdrowych donoszonych noworodków i z krwi obwodowej 62 dorosłych osób z rozpoznaniem bliznowca.Wyniki: W grupie noworodków stwierdzono 2 heterozygoty TG (1%) i 198 homozygot typu dzikiego TT (99%). W grupie osób dorosłych z keloidem stwierdzono jedynie homozygoty TT. Rozkład genotypów HYAL2 był zgodny z przewidywaną równowagą Hardy’ego–Weinberga.Wnioski: Pochodząca od człowieka neandertalskiego transwersja g.50318299T&gt;G (rs35455589) genu HYAL2, rzadko obecna w genomie współczesnych Polaków, nie jest związana z predyspozycją do bliznowca w tej populacji.

Mutations in HYAL2, Encoding Hyaluronidase 2, Cause a Syndrome of Orofacial Clefting and Cor Triatriatum Sinister in Humans and Mice.

Orofacial clefting is amongst the most common of birth defects, with both genetic and environmental components. Although numerous studies have been undertaken to investigate the complexities of the genetic etiology of this heterogeneous condition, this factor remains incompletely understood. Here, we describe mutations in the HYAL2 gene as a cause of syndromic orofacial clefting. HYAL2, encoding hyaluronidase 2, degrades extracellular hyaluronan, a critical component of the developing heart and palatal shelf matrix. Transfection assays demonstrated that the gene mutations destabilize the molecule, dramatically reducing HYAL2 protein levels. Consistent with the clinical presentation in affected individuals, investigations of Hyal2-/- mice revealed craniofacial abnormalities, including submucosal cleft palate. In addition, cor triatriatum sinister and hearing loss, identified in a proportion of Hyal2-/- mice, were also found as incompletely penetrant features in affected humans. Taken together our findings identify a new genetic cause of orofacial clefting in humans and mice, and define the first molecular cause of human cor triatriatum sinister, illustrating the fundamental importance of HYAL2 and hyaluronan turnover for normal human and mouse development.

Proximal and distal regulation of the HYAL1 gene cluster by the estrogen receptor α in breast cancer cells.

Chromosomal and genome abnormalities at the 3p21.3 locus are frequent events linked to epithelial cancers, including ovarian and breast cancers. Genes encoded in the 3p21.3 cluster include HYAL1, HYAL2 and HYAL3 members of hyaluronidases involved in the breakdown of hyaluronan, an abundant component of the vertebrate extracellular matrix. However, the transcriptional regulation of HYAL genes is poorly defined. Here, we identified the estrogen receptor ERα as a negative regulator of HYAL1 expression in breast cancer cells. Integrative data mining using METABRIC dataset revealed a significant inverse correlation between ERα and HYAL1 gene expression in human breast tumors. ChIP-Seq analysis identified several ERα binding sites within the 3p21.3 locus, supporting the role of estrogen as an upstream signal that diversely regulates the expression of 3p21.3 genes at both proximal and distal locations. Of these, HYAL1 was repressed by estrogen through ERα binding to a consensus estrogen response element (ERE) located in the proximal promoter of HYAL1 and flanked by an Sp1 binding site, required to achieve optimal estrogen repression. The repressive chromatin mark H3K27me3 was increased at the proximal HYAL1 ERE but not at other EREs contained in the cluster, providing a mechanism to selectively downregulate HYAL1. The HYAL1 repression was also specific to ERα and not to ERβ, whose expression did not correlate with HYAL1 in human breast tumors. This study identifies HYAL1 as an ERα target gene and provides a functional framework for the direct effect of estrogen on 3p21.3 genes in breast cancer cells.

Sunlight Adaptation Region of Neanderthal Genome Found in up to 65% of Modern East Asian Populations

With the Neanderthal genome now published, for the first time, scientists have a rich new resource of comparative evolution. For example, recently, scientists have shown that humans and Neanderthals once interbreed, with the accumulation of elements of Neanderthal DNA found in up to 5% in modern humans. Are there any advantages to the retention of Neanderthal DNA that favors modern humans? In a new article published in Molecular Biology and Evolution, Ding et al. (2013) present evidence for the accumulation of a Neanderthal DNA region found on chromosome 3 that contains 18 genes, with several related to ultraviolet (UV)-light adaptation, including the Hyal2 gene. Their results reveal that this region was positively selected and enriched in East Asians, ranging from up to 49% in Japanese to 66% in southern Chinese. Interestingly, the authors note the geographic distribution of the Neanderthal genomic region suggests that UV-light mutations were shown to be lost during the exodus of modern human from Africa and reintroduced to Eurasians from Neanderthals. “Overall, it is still very controversial whether there is more Neanderthal DNA contributions to Asians than Europeans, as we have evidence to argue against this,” said Lin. “Although in the case of the Hyal2 variant, it did indeed have a higher frequency in Asians.” From 45,000 to 5,000 years BP, effective population sizes of the Neanderthal region increased at a steady rate. Notably, the growth rate of the effective population size increased at around 5,000–3,500 years BP, which suggests a population expansion event. This Asian-specific Neanderthal evolutionary event is also consistent with previous reports of higher levels of Neanderthal ancestry in East Asians than in Europeans.

Effects of Payena dasyphylla (Miq.) on hyaluronidase enzyme activity and metalloproteinases protein expressions in interleukin-1β stimulated human chondrocytes cells.

BackgroundHyaluronidases have been found as the target enzymes in the development of osteoarthritis (OA) disease. While there is still no curative treatment for this disease, recent studies on the treatment of OA were focused on the effectiveness of natural products which are expected to improve the symptoms with minimal side effects. The aim of this study was to screen selected Malaysian plants on their anti-hyaluronidase activity as well as to evaluate the active plant and its derived fractions on its potential anti-arthritic and antioxidant activities.MethodsA total of 20 methanolic crude extracts (bark and leaf) from ten different plants were screened using a colorimetric hyaluronidase enzymatic assay. The active plant extract (Payena dasyphylla) was then studied for its hyaluronidase inhibitory activity in the interleukin-1β (IL-1β) stimulated human chondrocytes cell line (NHAC-kn) using zymography method. The Payena dasyphylla methanolic bark extract was then fractionated into several fractions in where the ethyl acetate (EA) fraction was evaluated for its inhibitory effects on the HYAL1 and HYAL2 gene expressions using reverse transcription-polymerase chain reaction (RT-PCR) technique. While the MMP-3 and MMP-13 protein expressions were evaluated using western blot method. The phenolic and flavonoid contents of the three fractions as well as the antioxidant property of the EA fraction were also evaluated.ResultsBark extract of Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 91.63%. The plant extract also inhibited hyaluronidase expression in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml). Similarly, treatment with Payena dasyphylla ethyl acetate (EA) fraction (100 μg/ml) inhibited the HYAL1 and HYAL2 mRNA gene expressions as well as MMP-3 and MMP-13 protein expression in a dose dependent manner. Payena dasyphylla EA fraction has demonstrated the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively as compared to water and hexane fractions. In addition, the Payena dasyphylla EA fraction showed strong antioxidant activity with IC50 value of 11.64 ± 1.69 μg/mL.ConclusionThese findings have shown that Payena dasyphylla might contained potential phenolic compounds that inhibiting the key enzyme in osteoarthritis development, which is the hyaluronidase enzyme through interruption of HYAL1 and HYAL1 gene expressions. The degradation of cartilage could also be inhibited by the plant through suppression of MMP-3 and MMP-13 protein expressions. We also reported that the inhibitory effect of Payena dasyphylla on hyaluronidase activity and expression might be due to its anti-oxidant property.

Subtype Specific Elevated Expression of Hyaluronidase-1 (HYAL-1) in Epithelial Ovarian Cancer

BackgroundEpithelial ovarian cancer (EOC) is morphologically heterogeneous being classified as serous, endometrioid, clear cell, or mucinous. Molecular genetic analysis has suggested a role for tumor suppressor genes located at chromosome 3p in serous EOC pathogenesis. Our objective was to evaluate the expression of HYAL1, located at chromosome 3p21.3, in these EOC subtypes, and to investigate its correlation with the expression of steroid hormone receptors.Methodology/Principal FindingsWe determined the mRNA expression of HYAL1, estrogen receptor (ER)-α, ERβ and progesterone receptor (PR) in EOC tumor samples and cell lines using quantitative RT-PCR. We also examined the expression of these genes in a publicly available microarray dataset. HYAL-1 enzyme activity was measured in EOC cell lines and in plasma samples from patients. We found that HYAL1 mRNA expression was elevated in clear cell and mucinous EOC tissue samples, but not in serous and endometrioid samples, normal ovaries or benign tumors. Similar results were obtained by two different techniques and with tissue sample cohorts from two independent institutions. Concordantly, HYAL1 mRNA levels and enzymatic activity were elevated only in EOC cell lines derived from clear cell and mucinous subtypes. We also showed that HYAL1 mRNA was inversely correlated to that of ERα specifically in clear cell and mucinous EOCs. Additionally, ectopic expression of ERα in a clear cell EOC cell line (ER- and PR-negative) induced 50% reduction of HYAL1 mRNA expression, supporting a role of ERα in HYAL1 gene regulation. Significantly, HYAL-1 activity was also high in the plasma of patients with these EOC subtypes.Conclusions/SignificanceThis is the first report showing high HYAL-1 levels in EOC and demonstrating HYAL1 gene repression by ERα. Our results identify Hyaluronidase-1 as a potential target/biomarker for clear cell and mucinous EOCs and especially in tumors with low ERα levels.

Reactive Oxygen Species and Hyaluronidase 2 Regulate Airway Epithelial Hyaluronan Fragmentation

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALI), treated with PI-specific phospholipase C (PI-PLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported that HA degradation was mediated by reactive oxygen species (ROS) in human airways. Here we show that ROS increase Hyal2 expression and activity in NHBE cells and that the p38MAPK signaling pathway is involved in this effect. Hyal2 induction was confirmed by using small interfering RNA (siRNA) expressing lentivirus. These in vitro findings correlated in vivo with smokers, where increased Hyal2 immunoreactivity in the epithelium was associated with augmented levels of HA and the appearance of low molecular mass HA species in bronchial secretions. In summary, this work provides evidence that ROS induce Hyal2, suggesting that Hyal2 is likely responsible for the sustained HA fragmentation in the airway lumen observed in inflammatory conditions associated with oxidative stress.

Design and Evaluation of Three Pair Primers for Exon 1 Amplification of Hyaluroglucosaminidase-1 Gene

Problem statement: Hyaluronidase is an enzyme which catalyze hydrolysis of Hyaluronan (HA). Hyaluronan is important in cell migration during embryonic development, cellular proliferation and differentiation and has a structural role in connective tissues. Hyaluronidase deficiency is correlated with mucopolysaccharidosis IX. In human, hyaluronidase is encoded by HYAL1 gene. The mutation study of HYAL1 gene was carried out by many researchers, but until now, mutation study of HYAL1 still in progress and limited due to the lack of primer used in amplification of selected DNA sequence of HYAL1 gene and maximum length limitation imposed by DNA sequencer. Approach: The search for three pairs of primers for human exon 1 segmented amplification of HYAL1 gene was conducted and evaluated. The first step was to acquire HYAL1 gene sequence and then had it aligned with human chromosome 3 genomic contig sequence. Exon 1 of HYAL1 gene was found to be located at nt 201-1711 of the acquired human chromosome 3 genomic contig. Online Primer3 program was used to design three pairs of primers. The selected pairs of primer had been subjected to BLASTn operation for selectivity examination while onlineNetPrimer operation was carried out for examination of secondary structures. Results: The search for primers to amplify three different fragments of exon 1 of HYAL1 gene yielded three selected pairs of primers, namely forward primer 5’-TGACCCCCTACAAAAGCTCA-3’ (20 bp) and reverse primer 5’-AAGTCTCCGATTCCCCCACT-3’ (20 bp) for amplifying nt 1-551 of HYAL1 gene, forward primer 5’-AGTCCTGTGGGAGATGGCAGA-3’ (21 bp) and reverse primer 5’-CGGTAAATGTCCTTGGTGTCC-3’ (21 bp) for amplifying nt 355-1053 of HYAL1 gene and forward primer 5’-GCCATACCTGCTCCTGACTT-3’ (20 bp) and reverse primer 5’-ACAAGGTGGGCAGGTTACAG-3’ (20 bp) for amplifying nt 956-1511 of HYAL1 gene. When using these primers, nt 1-46 of amplified product of the first pair of primers and nt 555-584 of amplified product of the third pair of primers must not be considered because those are not part of exon 1 of HYAL1 gene. The results from both operations and trial to real samples using these primers indicated that all three pairs of primer were satisfactory for use. Conclusion/Recommendations: The three pairs of primers could be used to amplify specified segments of HYAL1 gene.

Hyaluronidase-1 (Hyal-1) Is Differentially Expressed in Epithelial Ovarian Cancer of Distinct Histopathological Subtypes.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic cancer in most Western countries. Because of its asymptomatic growth and the lack of effective screening methods, about 70% of all cases are diagnosed in an advanced stage. Although most of the patients respond to chemotherapy initially, recurrence rate is very high resulting in poor prognosis. Furthermore, EOCs are morphologically heterogeneous, and different histopathological subtypes have distinct molecular characteristics and diverse response to treatment. In the present work we investigated the expression pattern of hyaluronidase-1 (Hyal-1) in different histopathological EOC subtypes as well as in EOC cell lines. The HYAL1 gene (together with those of HYAL2 and HYAL3) is located on the short arm of chromosome 3 (3p), a region described to contain tumour suppressor genes implicated in lung and ovarian cancer pathogenesis. However, levels of both Hyal-1 and its substrate, hyaluronic acid (hyaluronan), have also been reported to be increased in bladder, prostate and head and neck cancers, and to be implicated in tumor progression and metastasis. However, up to date, no studies have been conducted to analyze the transcriptional status of this enzyme and its role in EOC. Herein, quantitative RT-PCR was performed in ovarian tumor samples classified as borderline, serous, endometrioid, mucinous or clear cell. Distinct grade and stage were also attributed according to the International Federation of Gynecology and Obstetrics (FIGO) criteria. Cell lines derived from different EOC subtypes or from normal ovarian epithelial cells (NOSE) were also included in these analyses. Our results show that Hyal-1 expression was significantly increased in clear cell and mucinous EOC samples when compared to borderline tumors. In contrast, lower levels of Hyal-1 expression were observed in serous samples, and no difference was observed between endometrioid and borderline tumors. Accordingly, higher mRNA expression levels and higher enzymatic activity were observed in EOC cell lines derived from clear cell tumors. Cell lines derived from serous tumors had no detectable enzymatic activity and low levels of mRNA expression. However, treatment of one of these cell lines with 5-Azacytidine (DNA methyltransferase inhibitor) restored its Hyal-1 expression and activity, indicating that transcription repression through epigenetic regulation is occurring in serous EOCs. No correlation could be made between Hyal-1 expression and ovarian tumor grade or stage. In conclusion, our results show that Hyal-1 is differentially expressed in distinct EOC subtypes, with high expression levels in mucinous and clear cell carcinomas and low in serous adenocarcinoma. It is important to mention that specificity and sensitivity of the CA-125 antigen is particularly not good for these two former EOC subtypes. Therefore, Hyal-1 could be a potential marker for these tumors; however, further validation using a bigger tissue/serum sample cohort is needed. Supported by NSERC, RRCancer and FRSQ. (poster)

Changes in expression of Hyaluronidase genes during immunization-induced IgE formation

Hyaluronan (HA) is a glycosaminoglycan that associates with various molecules to assemble a specific HA-rich extracellular matrix. Low and intermediate-molecular-weight HA induces cytokines, chemokines, and other mediators of inflammation and extracellular matrix (ECM) degradation. Hyaluronidases are a group of enzymes that degrade HA. As a model of patients with allergic diseases, we used egg albumin to induce high levels of IgE in rats and examined changes in the expression of hyaluronidase genes (Hyal1 and Hyal2) using reverse transcription-polymerase chain reaction (RT-PCR). The expression of Hyal2 was not affected by the overproduction of IgE induced with egg albumin, whereas the expression of the Hyal1 gene was suppressed. High-molecular-weight HA might be degraded by Hyal2 into intermediate-molecular-weight HA, which can trigger inflammation.

Characterization of Promoter Elements of the Human HYAL-2 Gene

The regulated catabolism of hyaluronan is critical to the function of many connective tissues. In cartilage, hyaluronan catabolism occurs locally by resident chondrocytes. To determine whether the expression of lysosomal hyaluronidases contributes to this regulation, the promoter elements associated with HYAL-2 gene expression were characterized. Human articular chondrocytes were found to express all three lysosomal hyaluronidases, HYAL-1, HYAL-2, and HYAL-3. HYAL-2 was the predominant gene product. Using 5' RACE (rapid amplification of cDNA ends) analysis, multiple transcription initiation sites were identified including a novel initiation site located within intron 1 of the gene expressed by human articular chondrocytes. The presence of multiple transcriptional initiation sites is a typical feature of TATA-less promoter regions, such as those of HYAL-2. Approximately 4000 bp of 5' flanking sequence of the HYAL-2 gene was characterized. Transient transfection of C-28/I2 cells with various 5' deletion constructs indicated that the region between +959 to +1158 (within intron 1) contains the basal promoter for HYAL-2 in chondrocytes. In addition, the region +224 to +958 contained a negative modulator that could control the basal expression level of HYAL-2. Treatment of human articular chondrocytes or C-28/I2 cells with various catabolic cytokines did not alter HYAL-2 mRNA expression, luciferase promoter expression, or hyaluronidase enzymatic activity. Thus, in chondrocytes HYAL-2 appears to be constitutively expressed and not inducibly regulated by catabolic agents. As such, it appears that the expression of lysosomal hyaluronidase participates little in the overall regulation of hyaluronan catabolism.

Expression of HYAL2 mRNA, hyaluronan and hyaluronidase in B-cell non-Hodgkin lymphoma: relationship with tumor aggressiveness.

Hyaluronidases and their substrate, hyaluronan (HA), were mainly explored in solid tumors but rarely in hematologic malignancies. While HA involvement was demonstrated in invasion and metastasis in most cases of solid tumors, the role of hyaluronidases in cancer progression remains controversial. One of the hyaluronidases, HYAL2, is suspected to be involved in the first step of HA degradation. In this work, HYAL2 mRNA, HA and total hyaluronidases expression were examined in lymphoma tissue extracts and correlated to the lymphoma subtype. Real-time RT-PCR was performed to evaluate HYAL2 mRNA. HA and hyaluronidase were assayed by enzyme-linked sorbent assay. Our results showed that HYAL2 mRNA expression was correlated to lymphoma diagnosis (p = 6 x 10(-3)) and was significantly lower in high-grade lymphoma, i.e., diffuse large B-cell diffuse lymphomas (DLBCLs). Several forms of hyaluronidase were detected by zymography and total hyaluronidase activity detected in tissue extracts was not significantly different according to tumor grade. HA levels also correlated to lymphoma subtype (p = 1 x 10(-5)) and were higher in DLBCLs. Moreover, HYAL2 mRNA and HA expressions were inversely correlated (p = 0.035). HYAL2 gene is localized on chromosome 3p21, which contains candidates tumor suppressor genes. Our results suggest that HYAL2 may have a prognostic significance in lymphomas and an antioncogenic activity. Conversely, HA overexpression in high-grade lymphomas is in favor of its involvement in tumor development and could provide a useful target for lymphoma therapy using HA-binding peptides.

Expression and regulation patterns of hyaluronidases in small cell lung cancer and glioma lines.

Hyaluronan and hyaluronidases have been proposed to be involved in tumor angiogenesis and invasion. Three hyaluronidases, HYAL1, HYAL2 and HYAL3, are located at the chromosomal region 3p21. In most small cell lung cancer (SCLC) lines the 3p21 region is part of a homozygote or heterozygote deletion. Gliomas are known to exist in a hyaluronan rich environment and express high levels of the hyaluronan receptor CD44. In a panel of SCLC and glioma cell lines the expression of HYAL1, HYAL2 and HYAL3 mRNA was examined. It was observed that the cell lines differed in their ability to splice out a retained intron in the 5' UTR of HYAL1 mRNA. A correlation seems to exist between the ability to splice out the retained 5' end intron of HYAL1 mRNA and the general hyaluronidase activity. In one cell line a substantial part of the hyaluronidase activity was abolished by immunoprecipitation of Hyal1, which strongly indicates that Hyal1 is the principal hyaluornidase in the examined cell lines. During severe hypoxia a significant reduction in both hyaluronidase mRNA and protein activity was found. These results support the theory of involvement of hyaluronidase in the angiogenic and invasive front of tumors.

JNK1 Physically Interacts with WW Domain-containing Oxidoreductase (WOX1) and Inhibits WOX1-mediated Apoptosis

Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53. Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis. Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1 prevention of cell cycle progression. By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33). The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation. Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1. A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation. This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis. Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.

Assignment of the porcine FUS1 gene to SSC13q21→q22 by somatic cell and radiation hybrid panel mapping

FUS1 is a candidate tumor suppressor gene organized in a 370 kb region which contains a set of 19 genes. The human FUS1 genomic sequence spans 3.3 kb containing 3 exons (NM_007275; Lerman and Minna, 2000). Overexpression of the FUS1 gene leads to G1 arrest and growth inhibition of lung cancer cells (Kondo et al., 2001). The human and the murine FUS1 genes were assigned to chromosome HSA3p21.3 and MMU9F1, respectively. The sequence characterization of a porcine PAC clone revealed the identical gene cluster for pigs and human with respect to genes FUS1–HYAL2–HYAL1– FUS2–HYAL3. The porcine HYAL3 gene has recently been mapped to SSC13q21→q22 by fluorescence in situ hybridization (FISH) and radiation hybrid panel analysis while the somatic cell hybrid panel analysis resulted in the localization of the gene to SSC13q42→ (1/2q46) (Gatphayak et al., 2003). Considering the gene order in the cluster, the latter assignment is in accordance with the mapping of the porcine HYAL1 gene – which was provisionally called LUCA1 (for LUng CAncer) – to SSC13q23→ (1/2)q41 by Lahbib-Mansais et al. (1999) also using the somatic cell hybrid panel. Here we report the mapping of the porcine FUS1 gene by somatic cell and radiation hybrid panel and provide enhanced knowledge about the localization of the gene cluster. Methods and materials