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

<p>Abstract</p> <p>Background</p> <p>Hyaluronan 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 (<it>HAS1</it>, <it>HAS2 </it>and <it>HAS3</it>) and hyaluronidases (<it>HYAL1 </it>and <it>HYAL2</it>), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.</p> <p>Methods</p> <p>A 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.</p> <p>Results</p> <p>The mRNA levels of <it>HAS1-3 </it>were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, <it>HAS3 </it>mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of <it>HYAL1 </it>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. <it>HYAL2 </it>mRNA was also reduced in cancer (p = 0.02) and correlated with <it>HYAL1</it> (r = 0.8, p = 0.0001). There was an inverse correlation between <it>HYAL1 </it>mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001).</p> <p>Conclusion</p> <p>The results indicated that <it>HYAL1 </it>and <it>HYAL2 </it>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 <it>HAS</it>s were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.</p

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin,biotin bond. Implications for the molecular mechanism of unbinding of HA, hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA, hyaladherin complexes and HA-rich extracellular matrices

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

<p>Abstract</p> <p>Background</p> <p>Hyaluronan, 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 (<it>HAS1</it>, <it>HAS2 </it>and <it>HAS3</it>) and hyaluronidases (<it>HYAL1 </it>and <it>HYAL2</it>), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1–3 immunoreactivity.</p> <p>Methods</p> <p>Normal 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.</p> <p>Results</p> <p>The levels of <it>HAS2 </it>and <it>HAS3 </it>mRNA (<it>HAS1 </it>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 <it>HYAL1 </it>mRNA level was 69% lower in grade 3 serous ovarian cancers compared to normal ovaries (P = 0.01). The expression of <it>HYAL1</it>, but not <it>HYAL2</it>, significantly correlated with the enzymatic activity of tissue hyaluronidases (r = 0.5; P = 0.006). An inverse correlation was noted between <it>HYAL1 </it>mRNA and the intensity of hyaluronan staining of the corresponding tissue sections (r = -0.4; P = 0.025).</p> <p>Conclusion</p> <p>The results indicate that in serous epithelial ovarian malignancies <it>HAS </it>expression is not consistently elevated but <it>HYAL1 </it>expression is significantly reduced and correlates with the accumulation of hyaluronan. (233 words)</p

A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices

A rat epidermal keratinocyte (REK) line which exhibits histodifferentiation nearly identical to the native epidermis when cultured at an air–liquid interface was used to study the metabolism of hyaluronan, the major intercellular macromolecule present in basal and spinous cell layers. Two different support matrices were used: reconstituted collagen fibrils with and without a covering basal lamina previously deposited by canine kidney cells. REKs formed a stratified squamous, keratinized epithelium on both support matrices. Hyaluronan and its receptor, CD44, colocalized in the basal and spinous layers similar to their distribution in the native epidermis. Most (approximately 75%) of the hyaluronan was retained in the epithelium when a basal lamina was present while most (approximately 80%) diffused out of the epithelium in its absence. While REKs on the two matrices synthesized hyaluronan at essentially the same rate, catabolism of this macromolecule was much higher in the epithelium on the basal lamina (half-life approximately 1 day, similar to its half-life in native human epidermis). The formation of a true epidermal compartment in culture bounded by the cornified layer on the surface and the basal lamina subjacent to the basal cells provides a good model within which to study epidermal metabolism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42232/1/418-113-4-265_s004180000128.pd

Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44

Adhesive interactions involving CD44, the cell surface receptor for hyaluronan, underlie fundamental processes such as inflammatory leukocyte homing and tumor metastasis. Regulation of such events is critical and appears to be effected by changes in CD44 N-glycosylation that switch the receptor "on" or "off" under appropriate circumstances. How altered glycosylation influences binding of hyaluronan to the lectin-like Link module in CD44 is unclear, although evidence suggests additional flanking sequences peculiar to CD44 may be involved. Here we show using X-ray crystallography and NMR spectroscopy that these sequences form a lobular extension to the Link module, creating an enlarged HA binding domain and a formerly unidentified protein fold. Moreover, the disposition of key N-glycosylation sites reveals how specific sugar chains could alter both the affinity and avidity of CD44 HA binding. Our results provide the necessary structural framework for understanding the diverse functions of CD44 and developing novel therapeutic strategies

Integration of the activation of the human hyaluronan synthase 2 gene promoter by common cofactors of the transcription factors retinoic acid receptor and nuclear factor kappaB

Hyaluronan (HA) is a polysaccharide of the vertebrate extracellular matrix, produced by three related HA synthases (HASs) that influence numerous physiological processes. We screened the first 2250 bp of the HAS2 promoter for transcription factor response elements (REs) in silico and found 1 cluster of 2 retinoic acid (RA) REs, 3 discrete NF-kappaB factors, and 12 Sp1 REs. In parallel, we scanned nine overlapping promoter regions in HaCaT human immortalized keratinocytes using chromatin immunoprecipitation assays to identify binding of mediator, coactivator, and corepressor proteins and Sp1 transcription factor in response to all-trans-RA and tumor necrosis factor-alpha (TNF-alpha). We found that all-trans-RA modulated the binding of the RA receptor and several coregulators to the region containing the RARE cluster at position -1230. The importance of this region is supported in reporter gene assays by the all-trans-RA induction of the respective promoter region. Similarly, we showed by chromatin immunoprecipitation assays as well as by gel-shift assays with nuclear extracts that TNF-alpha induced NF-kappaB binding to regions at positions -380, -1420, and -1890, demonstrated its association with RNA polymerase II and cofactor proteins, and confirmed the functionality of the respective promoter regions in vivo. These findings partially explain the induction of HAS2 mRNA by all-trans-RA and TNF-alpha and provide an example how the action of different transcription factor families can use the same cofactors