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Background: Hyaluronan (HA), the main component of the extracellular matrix, is involved in tissue elasticity and cell scaffolding, and in progression of conditions such as cancer, inflammation and wound healing. Signaling by G protein coupled receptor (GPCR) activation increases expression of hyaluronan synthase (HAS) and HA production. The β2 adrenergic receptor (β2AR) is a catecholamine-liganded GPCR that is involved in cancer progression and wound healing. Since HA and β2AR are involved in a common pathology, we investigated whether β2AR signaling regulates HA production., Methods: After stimulating β2AR-expressing cells with a β agonist, the amount of HA in the culture medium was measured and HAS expression was examined by real-time PCR. A variety of signaling molecule inhibitors were used to identify signaling pathways that alter HAS expression., Results: β2AR activation increased HA production and enhanced HAS2 expression. The increase in HAS2 expression by β2AR activation occurred via the Gs - adenylyl cyclase - PKA - CREB signal transduction pathway., Conclusions: Downstream signal transduction by β2AR activation increased HA production by enhancing transcription of the HAS2 gene. This study suggests that β2AR is a GPCR that regulates HA production, and that stimulation with a catecholamine (β2 agonist) can regulate HA production., General Significance: β2AR may function through regulation of HA production in cancer progression and wound healing., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the skin, the metabolism of hyaluronan (HA) is highly regulated. Aging leads to chronic low-grade inflammation, which is characterized by elevated levels of pro-inflammatory cytokines; however, the relationship between inflammation and HA metabolism is not clear. Herein, we investigated the effects of a mixture of pro-inflammatory cytokines containing TNF-α, IL-1β, and IL-6 on HA metabolism in human skin fibroblasts. Treatment with the cytokine mixture for 24 h suppressed HA depolymerization via downregulation of HYBID (HA-binding protein involved in HA depolymerization/KIAA1199/CEMIP) and promoted HA synthesis via upregulation of HAS2 in human skin fibroblasts. Moreover, HAS2-dependent HA synthesis was driven mainly by IL-1β with partial contribution from TNF-α. Transmembrane protein 2 (TMEM2/CEMIP2), which was previously reported as a candidate hyaluronidase, was upregulated by the cytokine mixture, suggesting that TMEM2 might not function as a hyaluronidase in human skin fibroblasts. Furthermore, the effects of the cytokine mixture on HA metabolism were observed in fibroblasts after 8 days of treatment with cytokines during three passages. Thus, we have shown that HYBID-mediated HA metabolism is negatively regulated by the pro-inflammatory cytokine mixture, providing novel insights into the relationship between inflammation and HA metabolism in the skin., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare. The authors belong to a laboratory (Cosmetic Health Science) supported by Ichimaru Pharcos Co Ltd., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked β-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr 328 position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the casein kinase 1 substrate motif. These results suggest that Thr 328 phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The polysaccharide hyaluronan (HA) is abundant in pancreatic cancer (PC) tissue and promotes pancreatic cancer cell (PCC) motility in vitro. However, it is controversial as to whether high-molecular-weight HA (HMW-HA) or low-molecular weight HA(LMW-HA) is present in the pancreatic cancer stroma and whether PCC or pancreatic stellate cell (PSC) in PC tissue produces HA. We thereby aim to characterize the molecular weight and source of HA in PC tissue that promotes cancer cell motility., Methods: We analyzed the expression of hyaluronan synthase 2 (HAS2) and the hydrolyzing enzyme hyaluronidase 1 (HYAL1) in PCC lines and pancreatic stellate cells (PSCs) using real-time PCR. HA production in the supernatant of PCC lines and PSCs and in PC tissues was quantitatively and qualitatively examined. Finally, we knocked down HYAL1 expression in one of the PCC line PANC-1 cells and analyzed the impact on cell migration., Results: HAS2 was abundantly expressed in activated PSCs (aPSCs) but less so in quiescent PSCs (qPSCs) and PCC lines. The baseline expression of HYAL1 did not differ among the cell types. The concentration of HMW-HA was higher in the supernatant of aPSCs than in that of PCC lines. Treatment with exogenous HMW-HA promoted PANC-1 cell motility. Knockdown of HYAL1 decreased HMW-HA-promoted PANC-1 cell migration, which was accompanied by a decrease in intracellular HA levels., Conclusion: aPSCs are an important source of stromal HMW-HA, which promotes PCC migration in an HYAL1-dependent manner in PC., (Copyright © 2019 Elsevier Inc. All rights reserved.)