Tumor microenvironment and breast cancer survival: combined effects of breast fat, M2 macrophages and hyaluronan create a dismal prognosis.

PURPOSE: Tumor microenvironment, including inflammatory cells, adipocytes and extracellular matrix constituents such as hyaluronan (HA), impacts on cancer progression. Systemic metabolism also influences tumor growth e.g. obesity and type 2 diabetes (T2D) are risk factors for breast cancer. Here, in 262 breast cancer cases, we explored the combined impacts on survival of M2-like tumor associated macrophages (TAMs), the abundance of breast fat visualized as low density in mammograms, and tumor HA, and their associations with T2D. METHODS: Mammographic densities were assessed visually from the diagnostic images and dichotomized into very low density (VLD, density ≤ 10%, "fatty breast") and mixed density (MID, density > 10%). The amounts of TAMs (CD163+ and CD68+) and tumor HA were determined by immunohistochemistry. The data of T2D was collected from the patient records. Statistical differences between the parameters were calculated with Chi square or Mann-Whitney test and survival analyses with Cox's model. RESULTS: A combination of fatty breasts (VLD), abundance of M2-like TAMs (CD163+) and tumor HA associated with poor survival, as survival was 88-89% in the absence of these factors but only 40-47% when all three factors were present (p < 0.001). Also, an association between T2D and fatty breasts was found (p < 0.01). Furthermore, tumors in fatty breasts contained more frequently high levels of M2-like TAMs than tumors in MID breasts (p = 0.01). CONCLUSIONS: Our results demonstrate a dramatic effect of the tumor microenvironment on breast cancer progression. We hypothesize that T2D as well as obesity increase the fat content of the breasts, subsequently enhancing local pro-tumoral inflammation.

Peritumoral ADC values in breast cancer: region of interest selection, associations with hyaluronan intensity, and prognostic significance.

OBJECTIVES: We aimed to evaluate the differences in peritumoral apparent diffusion coefficient (ADC) values by four different ROI selection methods and to validate the optimal method. Furthermore, we aimed to evaluate if the peritumor-tumor ADC ratios are correlated with axillary lymph node positivity and hyaluronan accumulation. METHODS: Altogether, 22 breast cancer patients underwent 3.0-T breast MRI, histopathological evaluation, and hyaluronan assay. Paired t and Friedman tests were used to compare minimum, mean, and maximum values of tumoral and peritumoral ADC by four methods: (M1) band ROI, (M2) whole tumor surrounding ROI, (M3) clockwise multiple ROI, and (M4) visual assessment of ROI selection. Subsequently, peritumor/tumor ADC ratios were compared with hyaluronan levels and axillary lymph node status by the Mann-Whitney U test. RESULTS: No statistically significant differences were found among the four ROI selection methods regarding minimum, mean, or maximum values of tumoral and peritumoral ADC. Visual assessment ROI measurements represented the less time-consuming evaluation method for the peritumoral area, and with sufficient accuracy. Peritumor/tumor ADC ratios obtained by all methods except the clockwise ROI (M3) showed a positive correlation with hyaluronan content (M1, p = 0.004; M2, p = 0.012; M3, p = 0.20; M4, p = 0.025) and lymph node metastasis (M1, p = 0.001; M2, p = 0.007; M3, p = 0.22; M4, p = 0.015), which are established factors for unfavorable prognosis. CONCLUSIONS: Our results suggest that the peritumor/tumor ADC ratio could be a readily applicable imaging index associated with axillary lymph node metastasis and extensive hyaluronan accumulation. It could be related to the biological aggressiveness of breast cancer and therefore might serve as an additional prognostic factor. KEY POINTS: • Out of four different ROI selection methods for peritumoral ADC evaluation, measurements based on visual assessment provided sufficient accuracy and were the less time-consuming method. • The peritumor/tumor ADC ratio can provide an easily applicable supplementary imaging index for breast cancer assessment. • A higher peritumor/tumor ADC ratio was associated with axillary lymph node metastasis and extensive hyaluronan accumulation and might serve as an additional prognostic factor.

Extracellular ATP activates hyaluronan synthase 2 (HAS2) in epidermal keratinocytes via P2Y2, Ca2+ signaling, and MAPK pathways.

Extracellular nucleotides are used as signaling molecules by several cell types. In epidermis, their release is triggered by insults such as ultraviolet radiation, barrier disruption, and tissue wounding, and by specific nerve terminals firing. Increased synthesis of hyaluronan, a ubiquitous extracellular matrix glycosaminoglycan, also occurs in response to stress, leading to the attractive hypothesis that nucleotide signaling and hyaluronan synthesis could also be linked. In HaCaT keratinocytes, ATP caused a rapid and strong but transient activation of hyaluronan synthase 2 (HAS2) expression via protein kinase C-, Ca2+/calmodulin-dependent protein kinase II-, mitogen-activated protein kinase-, and calcium response element-binding protein-dependent pathways by activating the purinergic P2Y2 receptor. Smaller but more persistent up-regulation of HAS3 and CD44, and delayed up-regulation of HAS1 were also observed. Accumulation of peri- and extracellular hyaluronan followed 4-6 h after stimulation, an effect further enhanced by the hyaluronan precursor glucosamine. AMP and adenosine, the degradation products of ATP, markedly inhibited HAS2 expression and, despite concomitant up-regulation of HAS1 and HAS3, inhibited hyaluronan synthesis. Functionally, ATP moderately increased cell migration, whereas AMP and adenosine had no effect. Our data highlight the strong influence of adenosinergic signaling on hyaluronan metabolism in human keratinocytes. Epidermal insults are associated with extracellular ATP release, as well as rapid up-regulation of HAS2/3, CD44, and hyaluronan synthesis, and we show here that the two phenomena are linked. Furthermore, as ATP is rapidly degraded, the opposite effects of its less phosphorylated derivatives facilitate a rapid shut-off of the hyaluronan response, providing a feedback mechanism to prevent excessive reactions when more persistent signals are absent.

Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 µm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

UDP-sugar substrates of HAS3 regulate its O-GlcNAcylation, intracellular traffic, extracellular shedding and correlate with melanoma progression.

Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.

Interleukin-1ß-induced Reduction of CD44 Ser-325 Phosphorylation in Human Epidermal Keratinocytes Promotes CD44 Homomeric Complexes, Binding to Ezrin, and Extended, Monocyte-adhesive Hyaluronan Coats.

The proinflammatory cytokine interleukin-1ß (IL-1ß) attracts leukocytes to sites of inflammation. One of the recruitment mechanisms involves the formation of extended, hyaluronan-rich pericellular coats on local fibroblasts, endothelial cells, and epithelial cells. In the present work, we studied how IL-1ß turns on the monocyte adhesion of the hyaluronan coat on human keratinocytes. IL-1ß did not influence hyaluronan synthesis or increase the amount of pericellular hyaluronan in these cells. Instead, we found that the increase in the hyaluronan-dependent monocyte binding was associated with the CD44 of the keratinocytes. Although IL-1ß caused a small increase in the total amount of CD44, a more marked impact was the decrease of CD44 phosphorylation at serine 325. At the same time, IL-1ß increased the association of CD44 with ezrin and complex formation of CD44 with itself. Treatment of keratinocyte cultures with KN93, an inhibitor of calmodulin kinase 2, known to phosphorylate Ser-325 in CD44, caused similar effects as IL-1ß (i.e. homomerization of CD44 and its association with ezrin) and resulted in increased monocyte binding to keratinocytes in a hyaluronan-dependent way. Overexpression of wild type CD44 standard form, but not a corresponding CD44 mutant mimicking the Ser-325-phosphorylated form, was able to induce monocyte binding to keratinocytes. In conclusion, treatment of human keratinocytes with IL-1ß changes the structure of their hyaluronan coat by influencing the amount, post-translational modification, and cytoskeletal association of CD44, thus enhancing monocyte retention on keratinocytes.

Fluorescence resonance energy transfer (FRET) and proximity ligation assays reveal functionally relevant homo- and heteromeric complexes among hyaluronan synthases HAS1, HAS2, and HAS3.

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

Hexosamine biosynthesis in keratinocytes: roles of GFAT and GNPDA enzymes in the maintenance of UDP-GlcNAc content and hyaluronan synthesis.

UDP-N-acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAc modification of intracellular proteins. However, little is known about the regulation of hexosamine biosynthesis that controls UDP-GlcNAc content. Four enzymes can catalyze the crucial starting point of the pathway, conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P): glutamine-fructose-6-phosphate aminotransferases (GFAT1 and 2) and glucosamine-6-phosphate deaminases (GNPDA1 and 2). Using siRNA silencing, we studied the contributions of these enzymes to UDP-GlcNAc content and hyaluronan synthesis in human keratinocytes. Depletion of GFAT1 reduced the cellular pool of UDP-GlcNAc and hyaluronan synthesis, while simultaneous blocking of both GNPDA1 and GDPDA2 exerted opposite effects, indicating that in standard culture conditions keratinocyte GNPDAs mainly catalyzed the reaction from GlcN6P back to Fru6P. However, when hexosamine biosynthesis was blocked by GFAT1 siRNA, the effect by GNPDAs was reversed, now catalyzing Fru6P towards GlcN6P, likely in an attempt to maintain UDP-GlcNAc content. Silencing of these enzymes also changed the gene expression of related enzymes: GNPDA1 siRNA induced GFAT2 which was hardly measurable in these cells under standard culture conditions, GNPDA2 siRNA increased GFAT1, and GFAT1 siRNA increased the expression of hyaluronan synthase 2 (HAS2). Silencing of GFAT1 stimulated GNPDA1 and GDPDA2, and inhibited cell migration. The multiple delicate adjustments of these reactions demonstrate the importance of hexosamine biosynthesis in cellular homeostasis, known to be deranged in diseases like diabetes and cancer.

High extent of O-GlcNAcylation in breast cancer cells correlates with the levels of HAS enzymes, accumulation of hyaluronan, and poor outcome.

PURPOSE: Obesity and oversupply of glucose, e.g., due to nutritional factors may shape the tumor microenvironment favorable for tumor progression. O-GlcNAcylation, a reversible modification of intracellular proteins, influences on several cellular functions and is connected to many diseases including cancer. Glycosaminoglycan hyaluronan (HA) enhances tumor progression and in breast cancer HA accumulation associates strongly with poor outcome. In vitro studies have suggested that O-GlcNAcylation may enhance HA synthesis. The aim of this study was to investigate the correlations between O-GlcNAcylation, HA-related parameters, and disease outcome in a clinical breast cancer material consisting of 278 breast cancer cases. METHODS: In microscopic analyses, O-GlcNAc staining of the breast carcinoma cells was evaluated in several randomly picked high-power fields of each section. The extent of cytoplasmic O-GlcNAc staining was graded as either low or high according to the intensity of the staining and the percentage of stained cells. The extent of nuclear O-GlcNAc staining was categorized as either low or high according to the percentage of stained nuclei. RESULTS: A high extent of both cytoplasmic and nuclear O-GlcNAcylation correlated with an increased relapse rate, development of distant metastases, and poor outcome. A high extent of cytoplasmic O-GlcNAcylation correlated also with the accumulation of all hyaluronan synthase (HAS1-3) proteins and with a large amount of HA in the tumor stroma. In addition, a high extent of nuclear O-GlcNAcylation associated with obesity. CONCLUSIONS: The results suggest a mechanistic association between increased O-GlcNAcylation and HA synthesis, leading to a HA-rich microenvironment favorable for breast cancer progression.

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion.

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells.

Extracellular UDP-glucose activates P2Y14 Receptor and Induces Signal Transducer and Activator of Transcription 3 (STAT3) Tyr705 phosphorylation and binding to hyaluronan synthase 2 (HAS2) promoter, stimulating hyaluronan synthesis of keratinocytes.

Hyaluronan, a major matrix molecule in epidermis, is often increased by stimuli that enhance keratinocyte proliferation and migration. We found that small amounts of UDP-sugars were released from keratinocytes and that UDP-glucose (UDP-Glc) added into keratinocyte cultures induced a specific, rapid induction of hyaluronan synthase 2 (HAS2), and an increase of hyaluronan synthesis. The up-regulation of HAS2 was associated with JAK2 and ERK1/2 activation, and specific Tyr(705) phosphorylation of transcription factor STAT3. Inhibition of JAK2, STAT3, or Gi-coupled receptors blocked the induction of HAS2 expression by UDP-Glc, the latter inhibitor suggesting that the signaling was triggered by the UDP-sugar receptor P2Y14. Chromatin immunoprecipitations demonstrated increased promoter binding of Tyr(P)(705)-STAT3 at the time of HAS2 induction. Interestingly, at the same time Ser(P)(727)-STAT3 binding to its response element regions in the HAS2 promoter was unchanged or decreased. UDP-Glc also stimulated keratinocyte migration, proliferation, and IL-8 expression, supporting a notion that UDP-Glc signals for epidermal inflammation, enhanced hyaluronan synthesis as an integral part of it.

Rab10-mediated endocytosis of the hyaluronan synthase HAS3 regulates hyaluronan synthesis and cell adhesion to collagen.

Hyaluronan synthases (HAS1-3) are unique in that they are active only when located in the plasma membrane, where they extrude the growing hyaluronan (HA) directly into cell surface and extracellular space. Therefore, traffic of HAS to/from the plasma membrane is crucial for the synthesis of HA. In this study, we have identified Rab10 GTPase as the first protein known to be involved in the control of this traffic. Rab10 colocalized with HAS3 in intracellular vesicular structures and was co-immunoprecipitated with HAS3 from isolated endosomal vesicles. Rab10 silencing increased the plasma membrane residence of HAS3, resulting in a significant increase of HA secretion and an enlarged cell surface HA coat, whereas Rab10 overexpression suppressed HA synthesis. Rab10 silencing blocked the retrograde traffic of HAS3 from the plasma membrane to early endosomes. The cell surface HA coat impaired cell adhesion to type I collagen, as indicated by recovery of adhesion following hyaluronidase treatment. The data indicate a novel function for Rab10 in reducing cell surface HAS3, suppressing HA synthesis, and facilitating cell adhesion to type I collagen. These are processes important in tissue injury, inflammation, and malignant growth.

The reciprocal association between mammographic breast density, hyaluronan synthesis and patient outcome.

Low mammographic breast density (MBD) and increased hyaluronan (HA) synthesis have been shown to have adverse effects on breast cancer prognosis. We aimed at elucidating the background of risk associated with mammographic characteristics, MBD and HA and its synthesizing isoforms in an attempt to uncover potential underlying biological mechanisms. MBD and mammographic characteristics of 270 patients were classified according to percentile density (very low density VLD, ≤25 %; mixed density MID, >25 %) and the BI-RADS 5th edition lexicon. Breast density and mammographic features were correlated with the localization and expression of HA, CD44, and HAS1-3 isoforms, and their combined effect on patients' survivals was explored. VLD showed an increased level of HA-positive carcinoma cells and stromal HA, HAS2, and HAS3. Tumors presenting as masses had more HA-positive carcinoma cells and more stromal HAS2 and HAS3. Indistinct margin tumors showed more stromal HA and HAS3. Patients who combined both VLD breasts with either high HA in carcinoma cells or stroma showed a worse prognosis compared to low levels (carcinoma cells 58.0 vs. 80.5 %, p = 0.001; stroma 64.2 vs. 79.6 %, p = 0.017), while no similar HA-related effect was observed in MID breasts. Our findings suggest a strong reciprocal relationship between low MBD and HA expression and synthesis. The expression of both factors simultaneously leads to an especially adverse prognostic effect which might have an impact on treatment decision in the future. Moreover, HA around cancer cells may inhibit chemotherapy agents and antibody treatments from reaching cancer cells.

High numbers of macrophages, especially M2-like (CD163-positive), correlate with hyaluronan accumulation and poor outcome in breast cancer.

AIMS: High amounts of tumour-associated macrophages (TAMs) and hyaluronan (HA) correlate with tumour aggressiveness in breast cancer, but the relationship between these parameters is unclear. The aim of this study was to assay the numbers of TAMs in 278 human breast cancer cases, and their correlations with HA-related factors, clinical variables, and outcome. METHODS AND RESULTS: The immunoreactivities for CD163 and CD68 were considered as indicators for M2-like and all TAMs, respectively. The numbers of TAMs were counted in at least four hot spots, and averaged to represent the numbers of TAMs in each section. In the statistical analyses, the numbers were graded as either low or high according to the median. High numbers of TAMs correlated with a high tumour HA content, HA synthases, CD44 positivity, and poor outcome. The number of CD163-positive cells represented a strong independent prognostic factor. There was also a significant correlation between obesity and a high number of CD163-positive cells. CONCLUSIONS: Concurrent increases in TAMs and HA in breast cancer indicate that the accumulation of HA facilitates macrophage infiltration and inflammatory responses during human breast cancer progression.

Decreased hyaluronidase 1 expression is associated with early disease recurrence in human endometrial cancer.

OBJECTIVE: Hyaluronidases (HYAL1 and HYAL2) are key enzymes in the degradation of hyaluronan, and their expression has been altered in various cancer types. We previously showed that hyaluronan accumulation in endometrial carcinomas was correlated with decreased mRNA expression of the HYAL genes. In this study, we analyzed HYAL1 and HYAL2 protein expressions in normal and precancerous endometrial tissues and in endometrial carcinomas. We also investigated whether the protein levels were associated with clinicopathological factors, invasion, and disease recurrence. METHODS: A total of 343 tissue specimens from normal, atrophic, hypertrophic, and neoplastic endometria were analyzed immunohistochemically for HYAL1 and HYAL2 expressions. The results were correlated with clinicopathological factors, the expression of the epithelial-mesenchymal transition marker, E-cadherin, and disease recurrence. RESULTS: Reduced HYAL1 expression was associated with the progression of endometrial carcinomas towards higher grades and also with large tumor sizes, lymph node metastasis, and lymphovascular invasion. Reduced expression of both HYAL1 and HYAL2 was associated with deep myometrial invasion. HYAL2 expression was primarily constant in neoplastic tissues, but its expression was altered in different phases of the endometrial cycle. In addition, a reduction in HYAL1 expression was associated with the depletion of E-cadherin. In a multivariate analysis, reduced HYAL1 expression was an independent prognostic factor for early disease recurrence (HR 5.13, 95% CI: 1.131-23.270, p=0.034). CONCLUSIONS: This study showed that reduced HYAL1 expression was associated with endometrial carcinoma aggressiveness, which further supported the role of hyaluronan degradation in cancer progression.

Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1-3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.

Hyaluronan, a major epidermal extracellular matrix component, responds strongly to different kinds of injuries. This also occurs by UV radiation, but the mechanisms involved are poorly understood. The effects of a single ultraviolet B (UVB) exposure on hyaluronan content and molecular mass, and expression of genes involved in hyaluronan metabolism were defined in monolayer and differentiated, organotypic three-dimensional cultures of rat epidermal keratinocytes. The signals regulating the response were characterized using specific inhibitors and Western blotting. In monolayer cultures, UVB increased hyaluronan synthase Has1 mRNA already 4 h postexposure, with a return to control level by 24 h. In contrast, Has2 and Has3 were persistently elevated from 8 h onward. Silencing of Has2 and especially Has3 decreased the UVB-induced accumulation of hyaluronan. p38 and Ca(2+)/calmodulin-dependent protein kinase II pathways were found to be involved in the UVB-induced up-regulation of Has2 and Has3 expression, respectively, and their inhibition reduced hyaluronan deposition. However, the expressions of the hyaluronan-degrading enzymes Hyal1 and Hyal2 and the hyaluronan receptor Cd44 were also up-regulated by UVB. In organotypic cultures, UVB treatment also resulted in increased expression of both Has and Hyal genes and shifted hyaluronan toward a smaller size range. Histochemical stainings indicated localized losses of hyaluronan in the epidermis. The data show that exposure of keratinocytes to acute, low dose UVB increases hyaluronan synthesis via up-regulation of Has2 and Has3. The simultaneously enhanced catabolism of hyaluronan demonstrates the complexity of the UVB-induced changes. Nevertheless, enhanced hyaluronan metabolism is an important part of the adaptation of keratinocytes to radiation injury.

Hyaluronan synthase 1 (HAS1) requires higher cellular UDP-GlcNAc concentration than HAS2 and HAS3.

Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.

Hyaluronan synthases (HAS1-3) in stromal and malignant cells correlate with breast cancer grade and predict patient survival.

Accumulation of hyaluronan (HA) in pericellular stroma and carcinoma cells is predictive of unfavorable patient prognosis in many epithelial cancers. However, it is not known whether the HA originates from carcinoma or stromal cells, or whether increased expression of hyaluronan synthase proteins (HAS1-3) contributes to HA accumulation. In this study, localization and expression of HAS1-3 were evaluated immunohistochemically in 278 cases of human breast cancer, and correlated with prognostic factors and patient outcome. Both carcinoma cells and stromal cells were HAS-positive. In carcinoma cells, HAS1 and HA stainings correlated with each other, and HAS1 associated with estrogen receptor negativity, HER2 positivity, high relapse rate, and short overall survival. In stromal cells, the staining levels of all HAS isoforms correlated with the stromal HA staining, stromal cell CD44, high relapse rate, and short overall survival of the patients. In addition, expression levels of stromal HAS1 and HAS2 were related to obesity, large tumor size, lymph node positivity, and estrogen receptor negativity. Thus, stromal HAS1 and HAS3 were independent prognostic factors in the multivariate analysis. The data suggest that increased levels of HAS enzymes contribute to the accumulation of HA in breast cancer, and that HA is synthesized in carcinoma cells and stromal cells. The study also indicates that HAS enzyme levels are related to tumor aggressiveness and poor patient outcome representing potential targets for therapy.

Tissue distribution and subcellular localization of hyaluronan synthase isoenzymes.

Hyaluronan synthases (HAS) are unique plasma membrane glycosyltransferases secreting this glycosaminoglycan directly to the extracellular space. The three HAS isoenzymes (HAS1, HAS2, and HAS3) expressed in mammalian cells differ in their enzymatic properties and regulation by external stimuli, but clearly distinct functions have not been established. To overview the expression of different HAS isoenzymes during embryonic development and their subcellular localization, we immunostained mouse embryonic samples and cultured cells with HAS antibodies, correlating their distribution to hyaluronan staining. Their subcellular localization was further studied by GFP-HAS fusion proteins. Intense hyaluronan staining was observed throughout the development in the tissues of mesodermal origin, like heart and cartilages, but also for example during the maturation of kidneys and stratified epithelia. In general, staining for one or several HASs correlated with hyaluronan staining. The staining of HAS2 was most widespread, both spatially and temporally, correlating with hyaluronan staining especially in early mesenchymal tissues and heart. While epithelial cells were mostly negative for HASs, stratified epithelia became HAS positive during differentiation. All HAS isoenzymes showed cytoplasmic immunoreactivity, both in tissue sections and cultured cells, while plasma membrane staining was also detected, often in cellular extensions. HAS1 had brightest signal in Golgi, HAS3 in Golgi and microvillous protrusions, whereas most of the endogenous HAS2 immunoreactivity was localized in the ER. This differential pattern was also observed with transfected GFP-HASs. The large proportion of intracellular HASs suggests that HAS forms a reserve that is transported to the plasma membrane for rapid activation of hyaluronan synthesis.

Hyaluronan production enhances shedding of plasma membrane-derived microvesicles.

Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.