HYAL1 mRNA Research Articles

Abstract 5174: HYAL4: a new player in bladder cancer progression and diagnosis

Abstract BACKGROUND: Hyaluronidase (HAase) family of enzymes degrades hyaluronic acid (HA). In the human genome there are 6 HAase genes clustered on chromosomes 3p21.3 (HYAL-1, -2, -3) and 7q31.3 (HYAL-4, PH20, HYALP1). While PH20 and HYAL-2 are testicular and lysosomal HAases, HYAL1 is a tumor-derived HAase. HYAL1 promotes tumor growth, invasion, and angiogenesis and is a diagnostic and prognostic marker for bladder cancer (BCa). The expression of other HAases has not been investigated in either normal cells or tumor cells and tissues. In this study we examined the expression of all 6 HAases in bladder tissues, cells and urine. METHODS: Using time RT-PCR, we examined the mRNA levels of 6 HAase genes in BCa cell lines, 59 bladder tissues (normal (NBL) = 22; tumor (TBL) = 37) and 160 urine specimens (BCa = 52; normal = 18; history of BCa = 30; benign conditions = 59). The levels were normalized to β-actin. HYAL1 and HYAL4 were localized in bladder tissues by immunohistochemistry using anti-HYAL1 and anti-HYAL4 antibodies, respectively. The staining was graded for intensity (0 – 3) and area of staining. HYAL4 function was analyzed by siRNA transfection of HT1376 and HT5637 cell lines. RESULTS: HYAL1, HYAL4 and HYALP1 mRNA levels were significantly (6-13-fold) elevated in TBL tissues (12.5 ± 2.7; 29.7 ± 21.9; 26.7 ± 21.9) when compared to NBL tissues (1.2 ± 0.6; 2.2 ± 2.4; 4.2 ± 6.6; P 0.05). cDNA cloning revealed that HYALP1 is a pseudo gene that encodes a non-functional truncated protein. HYAL1 and HYAL4 staining was significantly elevated in TBL tissues (188 ± 57; 232 ± 91) when compared to NBL tissues (68.5 ± 21.3; 93 ± 46; P < 0.001). The staining for both HAases was exclusively localized in tumor cells. When compared to HYAL3, PH20, and HYAL2 mRNA levels, HYAL1, HYAL4 and HYALP1 mRNA levels were significantly elevated in BCa patients’ urine (P < 001). Urinary HYAL4 mRNA levels had higher sensitivity (78.9%) and specificity (86.2%) than HYAL1 mRNA levels (72.3%; 76.2%) to detect BCa. Both HAases detected high-grade BCa with 83% – 86% sensitivity. Down regulation of HYAL4 expression by siRNA decreased proliferation of HT1376 and HT5637 cells (2.3-fold) and induced apoptosis (2.6-fold). Conclusion: This is the first study showing that, like HYAL1, HYAL4 is a tumor derived HAase that is exclusively expressed in BCa cells. HYAL4 may be a potentially accurate marker for BCa diagnosis and may promote BCa growth and progression. Support: Grant NCI/NIH R01 CA-72821-09; Bankhead Coley Cancer Research Program Sylvester Comprehensive Cancer Center and CURED Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5174. doi:10.1158/1538-7445.AM2011-5174

Angiotensin converting enzyme inhibition blocks interstitial hyaluronan dissipation in the neonatal rat kidney via hyaluronan synthase 2 and hyaluronidase 1

A functional renin–angiotensin system (RAS) is required for normal kidney development. Neonatal inhibition of the RAS in rats results in long-term pathological renal phenotype and causes hyaluronan (HA), which is involved in morphogenesis and inflammation, to accumulate. To elucidate the mechanisms, intrarenal HA content was followed during neonatal completion of nephrogenesis with or without angiotensin converting enzyme inhibition (ACEI) together with mRNA expression of hyaluronan synthases (HAS), hyaluronidases (Hyal), urinary hyaluronidase activity and cortical lymphatic vessels, which facilitate the drainage of HA from the tissue. In 6–8days old control rats cortical HA content was high and reduced by 93% on days 10–21, reaching adult low levels. Medullary HA content was high on days 6–8 and then reduced by 85% to 12-fold above cortical levels at day 21. In neonatally ACEI-treated rats the reduction in HA was abolished. Temporal expression of HAS2 corresponded with the reduction in HA content in the normal kidney. In ACEI-treated animals cortical HAS2 remained twice the expression of controls. Medullary Hyal1 increased in controls but decreased in ACEI-treated animals. Urine hyaluronidase activity decreased with time in control animals while in ACEI-treated animals it was initially 50% lower and did not change over time. Cells expressing the lymphatic endothelial mucoprotein podoplanin in ACEI-treated animals were increased 18-fold compared to controls suggesting compensation. In conclusion, the high renal HA content is rapidly reduced due to reduced HAS2 and increased Hyal1 mRNA expressions. Normal angiotensin II function is crucial for inducing these changes. Due to the extreme water-attracting and pro-inflammatory properties of HA, accumulation in the neonatally ACEI-treated kidneys may partly explain the pathological renal phenotype of the adult kidney, which include reduced urinary concentration ability and tubulointerstitial inflammation.

Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma

BackgroundHyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.MethodsA total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry.ResultsThe mRNA levels of HAS1-3 were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, HAS3 mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of HYAL1 mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. HYAL2 mRNA was also reduced in cancer (p = 0.02) and correlated with HYAL1 (r = 0.8, p = 0.0001). There was an inverse correlation between HYAL1 mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001).ConclusionThe results indicated that HYAL1 and HYAL2 were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for HASs were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.

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.

Effects of Low-Intensity Pulsed Ultrasound on the Expression and Activity of Hyaluronan Synthase and Hyaluronidase in IL-1β-Stimulated Synovial Cells

The purpose of this study is to examine effects of low-intensity pulsed ultrasound (LIPUS) on metabolism of hyaluronan (HA) in synovial membrane cells stimulated by IL-1β. Rabbit knee synovial membrane cell line, HIG-82, was cultured in medium with the presence or absence of 1 ng/mL IL-1β, and after 4 h the cell was exposed to LIPUS for 15 min. The mRNA levels of HA synthase (HAS) 2,3, hyaluronidase (HYAL) 2, and cyclooxygenase (COX)-2 were examined by real-time PCR analysis. Concentrations of HA and PGE₂ were quantified by use of enzyme linked immunosorbent assay (ELISA). The COX-2 level was analyzed by western blotting. Gene levels of HAS2 and HAS3 in IL-1β-stimulated cells were up-regulated significantly (p < 0.01) by LIPUS. HYAL2 mRNA was up-regulated by the treatment with IL-1β, whereas down-regulated significantly (p < 0.01) by the following LIPUS exposure. Furthermore, IL-1β stimulation enhanced COX-2 and PGE₂ expression as compared to the untreated control, and IL-1β-induced COX-2 and PGE₂ expression was inhibited by LIPUS. These results suggest that LIPUS enhanced HA synthesis and inhibited HYAL2 expression, leading to the accumulation of high-molecular weight HA. Therefore, LIPUS stimulation may be a better candidate as medical remedy to treat inflammatory joint diseases accompanied with HA degradation in synovial fluid.

Modulation of Hyaluronan Fragmentation by Interleukin-1 Beta in Synovial Membrane Cells

Hyaluronan (HA) plays a crucial role in the lubricating and buffering properties of synovial fluid. The purpose of this study was to examine the effects of interleukin (IL)-1beta on HA degradation in cultured synovial membrane cells. The rabbit synovial membrane cell line HIG-82 was cultured with and without IL-1beta. The amounts of HA of varying molecular weights in the medium were analyzed using high-performance liquid chromatography, the mRNA levels of HA synthase (HAS) and hyaluronidase (HYAL) were analyzed by means of real-time PCR, and HYAL activity was analyzed by HA zymography. The amounts of HA with a molecular weight lower than 300 kDa, and between 300 and 1900 kDa, in the culture medium of HIG-82 cells were significantly higher in the presence of IL-1beta. However, the amount of HA with a molecular weight greater than 1900 kDa was significantly lower in the presence of IL-1beta. Both HAS2 and HAS3 mRNA levels were upregulated by treatment with IL-1beta. So, too, were the levels of HYAL1 and HYAL2 mRNA, which resulted in enhanced HYAL activity. However, HYAL activity was inhibited by transfection of HYAL2-siRNA. Our results suggest that IL-1beta is a crucial factor in the fragmentation of HA in inflammatory joints.

Modulation of hyaluronan catabolism in chondrocytes by mechanical stimuli

Hyaluronan (HA) is a component of the extracellular matrices of cartilage contributing to the structural and functional integrity. HA metabolism is regulated by both anabolic and catabolic processes; however, a great deal more of the detail has been unknown yet. The purpose of this study was to clarify the effect of excessive mechanical load on the expression and activity of hyaluronidase (HYAL) in chondrocytes with a special reference to the expressions of IL-1beta and tumor necrosis factor (TNF)-alpha. A cyclic tensile load of 22.8% cell elongation, regarded as an excessive mechanical stimulus, was applied to cultured rabbit knee articular chondrocytes. HYAL1, HYAL2, IL-1beta, and TNF-alpha mRNA levels were examined by quantitative real-time PCR analysis. The HYAL activity in culture medium was examined by HA zymography. Both HYAL1 and HYAL2 mRNA levels were upregulated significantly by the loading in cultured chondrocytes. HYAL activity was also enhanced as compared with unloaded controls. The IL-1beta mRNA level was upregulated significantly by the loading, and TNF-alpha mRNA level was slightly upregulated. HYAL1 and HYAL2 mRNA levels were upregulated significantly by IL-1beta treatment, resulting in a slight increase in HYAL activity. These results show that the expression of HYAL1 and HYAL2 in articular chondrocytes is enhanced by excessive mechanical stimuli and affected in part by induction of IL-1beta, leading to HA catabolism in articular cartilage.

Expression of Hyaluronan Synthases (HAS1–3) and Hyaluronidases (HYAL1–2) in Serous Ovarian Carcinomas: Inverse Correlation between HYAL1 and Hyaluronan Content

BackgroundHyaluronan, a tumor promoting extracellular matrix polysaccharide, is elevated in malignant epithelial ovarian tumors, and associates with an unfavorable prognosis. To explore possible contributors to the accumulation of hyaluronan, we examined the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1–3 immunoreactivity.MethodsNormal ovaries (n = 5) and 34 serous epithelial ovarian tumors, divided into 4 groups: malignant grades 1+2 (n = 10); malignant grade 3 (n = 10); borderline (n = 4) and benign epithelial tumors (n = 10), were analyzed for mRNA by real-time RT-PCR and compared to hyaluronidase activity, hyaluronan staining, and HAS1–3 immunoreactivity in tissue sections of the same specimens.ResultsThe levels of HAS2 and HAS3 mRNA (HAS1 was low or absent), were not consistently increased in the carcinomas, and were not significantly correlated with HAS protein or hyaluronan accumulation in individual samples. Instead, the median of HYAL1 mRNA level was 69% lower in grade 3 serous ovarian cancers compared to normal ovaries (P = 0.01). The expression of HYAL1, but not HYAL2, significantly correlated with the enzymatic activity of tissue hyaluronidases (r = 0.5; P = 0.006). An inverse correlation was noted between HYAL1 mRNA and the intensity of hyaluronan staining of the corresponding tissue sections (r = -0.4; P = 0.025).ConclusionThe results indicate that in serous epithelial ovarian malignancies HAS expression is not consistently elevated but HYAL1 expression is significantly reduced and correlates with the accumulation of hyaluronan. (233 words)

Effects of mechanical load on the expression and activity of hyaluronidase in cultured synovial membrane cells

Hyaluronan (HA) has lubricating and buffering functions in joints. Mechanical load is a regulatory factor of HA metabolism in joints, and HA synthesis by synovial membrane cells is modulated by mechanical load. However, the effects of mechanical load on HA catabolism by hyaluronidase (HYAL) in synovial membrane cells remain unclear. The purpose of this study was to evaluate the effects of cyclic tensile load on the expression and activity of HYAL in synovial membrane cells. A cyclic tensile load of 22.8% cell elongation was applied to cultured rabbit synovial membrane cells for 3 to 48 h with or without cycloheximide. HYAL1 and HYAL2 mRNA levels were evaluated by means of real-time polymerase chain reaction (PCR) analysis. HYAL activity in the cell culture was analyzed by means of HA zymography with or without HYAL2-small interfering (si) RNA. Levels of both HYAL1 and HYAL2 mRNA were up-regulated significantly (p < 0.01) by the cyclic tensile load with or without cycloheximide. HYAL activity was detected in the loaded cell cultures and was suppressed substantially by HYAL2-siRNA. HYAL activity was undetectable in unloaded cell cultures. These results show that a cyclic tensile load induces the expression and activity of HYAL in synovial membrane cells, suggesting that increased HYAL by mechanical load affects HA catabolism in synovial fluid.

Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer

Chromosomal and genome abnormalities of 3p are frequent events in many epithelial tumours, including lung cancer. Several critical regions with high frequency of hemi--and homozygous deletions in tumours were detected on 3p and more then 20 different cancer-related genes were identified in 3p21.3 locus. Real-time PCR was used to measure mRNA level of tumour-suppressor genes and candidates in 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1 and HYAL2 in basic types of non-small cell lung cancer (NSCLC)--squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). Significant (from 2 to 100 times) and frequent (from 44 to 100%) mRNA level decrease was shown in NSCLC. Level and frequency of mRNA decrease for all genes depended on histological type of NSCLC. Down-regulation of RASSF1A and ITGA9 was associated significantly with AC progression, the same tendency was found for genes RBSP3/CTDSPL, NPRL2/G21, HYAL1 and HYAL2. On the contrary, down-regulation of all genes in SCC was not associated with clinical stages, tumor cells differentiation and metastases in lymph nodes. Significant decrease of RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1 and HYAL2 mRNA levels (on average, 5-13 times) with high frequency (83-100%) was already shown at the first stage of SCC. Simultaneous decrease of all six genes mRNA level was found in the same tumor samples and was not depended on their localization on 3p21.3 and functions of the proteins. Spearman's correlation coefficient r(s) was from 0.63 to 0.91, P < 0.001. Co-regulation of gene pairs ITGA9 and HYAL2, HYAL1 and HYAL2, which mediate cell-cell adhesion and cell-matrix interaction, was suggested based on the obtained data. It was shown that genetic and epigenetic mechanisms were important for down-regulation of RBSP3/CTDSPL and ITGA9 genes. These results supported the hypothesis on simultaneous inactivation of cluster cancer-related genes in extended 3p21.3 locus during development and progression of lung cancer and other epithelial tumors. Significant and frequent decrease of mRNA level of six genes in SCC could be important for development of specific biomarker sets for early SCC diagnosis and new therapeutic approaches/strategies for NSCLC.

Mammalian Hyaluronidase Induces Ovarian Granulosa Cell Apoptosis and Is Involved in Follicular Atresia

During ovarian folliculogenesis, the vast majority of follicles will undergo atresia by apoptosis, allowing a few dominant follicles to mature. Mammalian hyaluronidases comprise a family of six to seven enzymes sharing the same catalytic domain responsible for hyaluronan hydrolysis. Interestingly, some of these enzymes have been shown to induce apoptosis. In the ovary, expression of three hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) has been documented. However, their precise cellular localization and role in ovarian regulation have not yet been defined. We herein investigated the possible involvement of these enzymes in ovarian atresia. First, we established a mouse model for ovarian atresia (gonadotropin withdrawal by anti-equine chorionic gonadotropin treatment) and showed that the mRNA levels of Hyal-1, Hyal-2, and Hyal-3 were significantly increased in apoptotic granulosa cells as well as in atretic follicles. Second, using ovaries of normally cycling mice, we demonstrated the correlation of Hyal-1 mRNA and protein expression with cleavage of caspase-3. In addition, we showed that expression of all three hyaluronidases induced apoptosis in transfected granulosa cells. Significantly, the induction of apoptosis by hyaluronidases was independent of catalytic activity, because enzymatically inactive Hyal-1 mutant (D157A/E159A) was as efficient as the wild-type enzyme in apoptosis induction. The activation of the extrinsic apoptotic signaling pathway was involved in this induction, because increased levels of cleaved caspase-8, caspase-3, and poly-ADP-ribose polymerase (PARP) were observed upon hyaluronidase ectopic expression. Our present findings provide a better understanding of the role of hyaluronidases in ovarian functions, showing for the first time their involvement in follicular atresia.

EVALUATION OF URINARY NUCLEAR MATRIX PROTEIN 22 TEST FOR DETECTION OF UPPER TRACT UROTHELIAL CARCINOMA

(87.2%, 74.4%) compared to the HAS1-HYAL1 (78.7%, 73.2%), HAS3HYAL1 (80.5%, 68.3%), HAS1-HAS2 (83%, 64.6%) and HAS2-HAS3 (85.1%, 67%) combinations. The HAS2-HYAL1 combination exhibited high sensitivity in detecting low(85.7%) and high-(87.8%) grade tumors. Five patients with a history of bladder cancer recurred within 6 months and 4 of them were positive for the HAS2HYAL1 and HAS2-HYAL1 combinations. CONCLUSIONS: A real time RT-PCR based assay for HYAL1 and HAS mRNA measurement detects bladder cancer with high

Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan (HA), a large polysaccharide abundant in the extracellular matrix of vertebrate tissues. Six genes have been predicted to encode hyaluronidases in humans, but the protein products of only SPAM1, HYAL1, and HYAL2 have been characterized. We have now expressed the mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in Baby Hamster Kidney (BHK) cells and demonstrated the presence of multiple forms of Hyal3 ranging from approximately 45 to 56 kDa in expression lysates. Complete and partial digestions of the expressed protein with PNGase F showed three N-linked oligosaccharides accounted for all forms of Hyal3 detected in expression lysates. Most of these oligosaccharides were Endo H sensitive, indicating that they were high mannose or hybrid N-linked oligosaccharides. Subcellular fractionation of Hyal3-expressing BHK cells by density gradient centrifugation revealed most Hyal3 in a low-density vesicular population. Low levels of Hyal3 were detected in higher density vesicles, but no colocalization with the late endosomal/lysosomal marker Lamp1 was found by immunofluorescence microscopy. BHK cells stably expressing Hyal3 had increased acid-active hyaluronidase activity, but no such activity was detected when Hyal3 was transfected into Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpression of Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels, suggesting that the increased hyaluronidase activity in these cells was due to Hyal1 rather than Hyal3. The results indicate that Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidase activity and that Hyal3 may contribute to HA metabolism by augmenting the activity of Hyal1.

Growth factor regulation of hyaluronan synthesis and degradation in human dermal fibroblasts: importance of hyaluronan for the mitogenic response of PDGF-BB

The glycosaminoglycan hyaluronan is important in many tissuerepair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to PDGF (platelet-derived growth factor)-BB and other growth factors. Human dermal fibroblasts exhibited the highest hyaluronan-synthesizing activity in response to PDGF-BB. Analysis of HAS (hyaluronan synthase) and HYAL (hyaluronidase) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (U0126) and for PI3K (phosphoinositide 3-kinase) (LY294002), as well as the SN50 inhibitor, which prevents translocation of the active NF-kappaB (nuclear factor kappaB) to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signalling pathways were without any significant effect. TGF-beta1 (transforming growth factor-beta1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Importantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hermes-1, inhibited PDGF-BB-stimulated [3H]thymidine incorporation of dermal fibroblasts. We conclude that the ERK MAPK and PI3K signalling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.

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.

Expression and activity of hyaluronidase in human periodontal ligament fibroblasts.

Hyaluronan is a major component of the extracellular matrix of periodontal ligament (PDL) contributing to the structural and functional integrity. Hyaluronans contribute to the buffering effect of the PDL during chewing, and they are also important in inflammation and wound healing. Hyaluronan is known to be synthesized and turned over by the resident PDL cells, although the mechanisms of hyaluronan metabolism still remain unclear. Hyaluronidase (HAase), an endoglycosidase, degrades hyaluronan into small fragments. Currently, 3 human HAases, HYAL1, HYAL2, and PH-20, have been identified and well characterized. This study was conducted to investigate the expression and activity of these HAases in cultured human PDL fibroblasts and to elucidate the mechanisms involved in hyaluronan metabolism under normal and inflammatory conditions. Human PDL fibroblasts derived from the periodontium of 3 premolars were cultured with or without interleukin (IL)-1beta (0.1 to 10 ng/ml) and tumor necrosis factor (TNF)-alpha (1 to 100 ng/ml) for 0 to 48 hours. The expression of HAase mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR, and the enzymatic activity was examined using hyaluronan zymography. PDL fibroblasts expressed HYAL1 and HYAL2 mRNAs, but not PH-20 mRNA. The expression of HYAL1 mRNA was enhanced by about 3.5- and 3.7-fold at maximum after 1-hour stimulation with 1 ng/ml IL-1beta and after 3-hour stimulation with 10 ng/ml TNF-alpha, respectively. The expression of HYAL2 and PH-20 mRNAs was not affected by stimulation with cytokines. HAase activity was detected in conditioned medium from PDL fibroblast cultures, and the activity was enhanced by treatment with 10 ng/ml TNF-alpha. These results suggest that PDL fibroblasts express HAases and generate HAase activity essential for extracellular hyaluronan metabolism under physiological and inflammatory conditions.

HYAL1LUCA-1, a candidate tumor suppressor gene on chromosome 3p21.3, is inactivated in head and neck squamous cell carcinomas by aberrant splicing of pre-mRNA

The hyaluronidase first isolated from human plasma, Hyal-1, is expressed in many somatic tissues. The Hyal-1 gene, HYAL1, also known as LUCA-1, maps to chromosome 3p21.3 within a candidate tumor suppressor gene locus defined by homozygous deletions and by functional tumor suppressor activity. Hemizygosity in this region occurs in many malignancies, including squamous cell carcinomas of the head and neck. We have investigated whether cell lines derived from such malignancies expressed Hyal-1 activity, using normal human keratinocytes as controls. Hyal-1 enzyme activity and protein were absent or markedly reduced in six of seven carcinoma cell lines examined. Comparative genomic and fluorescence in situ hybridization identified chromosomal deletions of one allele of HYAL1 in six of seven cell lines. Initial RT - PCR analyses demonstrated marked discrepancies between levels of HYAL1 mRNA and protein. Despite repeated sequence analyses, no mutations were found. However, two species of transcripts were identified when primers were used that included the 5' untranslated region. The predominant mRNA species did not correlate with protein translation and contained a retained intron. A second spliced form lacking this intron was found only in cell lines that produced Hyal-1 protein. Inactivation of HYAL1 in these tumor lines is a result of incomplete splicing of its pre-mRNA that appears to be epigenetic in nature. Oncogene (2000) 19, 870 - 877.

The Hyaluronidase Gene HYAL1 Maps to Chromosome 3p21.2–p21.3 in Human and 9F1–F2 in Mouse, a Conserved Candidate Tumor Suppressor Locus

We recently cloned and expressed the major hyaluronidase activity from human plasma, HYAL1, and found that the protein is 40% identical to the testicular hyaluronidase, PH-20. The HYAL1 mRNA sequence was used in a homology search of the mouse database of expressed sequence tags (dbEST). Two ESTs were obtained and, in combination with 5′RACE-PCR, were used to clone the mouse HYAL1 ortholog (Hyal1). Hyal1 codes for a protein of 462 amino acids that is 73% identical to the human sequence. Hyal1 stably expressed in human embryonic kidney cells resulted in a 20,000-fold increase of hyaluronidase activity. Sequence-tagged sites derived from the HYAL1 gene from both species were used to isolate P1 genomic clones that were used as probes for fluorescencein situhybridization. The human gene was localized to chromosome 3p21 and the mouse gene to a syntenic region on chromosome 9F1–F2. In mouse, serum hyaluronidase polymorphism has previously been mapped by an interspecific backcross to 60 cM from the centromere of chromosome 9, which corresponds to a cytogenetic location of 9F1–F2. The mouse Hyal1 gene is therefore very likely to be responsible for the hyaluronidase polymorphism linked to this locus. We also present evidence that human HYAL1 is identical to an uncharacterized gene positionally cloned by others from chromosome 3p21.3 that is homozygously deleted in several small-cell lung carcinoma cell lines.