Hyaluronan synthase 1: a mysterious enzyme with unexpected functions

Abstract

Hyaluronan synthase 1 (HAS1) is one of three isoenzymes responsible for cellular hyaluronan synthesis. Interest in HAS1 has been limited because its role in hyaluronan production seems to be insignificant compared to the two other isoenzymes, HAS2 and HAS3, which have higher enzymatic activity. Furthermore, in most cell types studied so far, the expression of its gene is low and the enzyme requires high concentrations of sugar precursors for hyaluronan synthesis, even when overexpressed in cell cultures. Both expression and activity of HAS1 are induced by pro-inflammatory factors like interleukins and cytokines, suggesting its involvement in inflammatory conditions. Has1 is upregulated in states associated with inflammation, like atherosclerosis, osteoarthritis, and infectious lung disease. In addition, both full length and splice variants of HAS1 are expressed in malignancies like bladder and prostate cancers, multiple myeloma, and malignant mesothelioma. Interestingly, immunostainings of tissue sections have demonstrated the role of HAS1 as a poor predictor in breast cancer, and is correlated with high relapse rate and short overall survival. Utilization of fluorescently tagged proteins has revealed the intracellular distribution pattern of HAS1, distinct from other isoenzymes. In all cell types studied so far, a high proportion of HAS1 is accumulated intracellularly, with a faint signal detected on the plasma membrane and its protrusions. Furthermore, the pericellular hyaluronan coat produced by HAS1 is usually thin without induction by inflammatory agents or glycemic stress and depends on CD44-HA interactions. These specific interactions regulate the organization of hyaluronan into a leukocyte recruiting matrix during inflammatory responses. Despite the apparently minor enzymatic activity of HAS1 under normal conditions, it may be an important factor under conditions associated with glycemic stress like metabolic syndrome, inflammation, and cancer.

Keywords: CD44; cancer; cytokines; hyaluronan; hyaluronan synthase; inflammation.

Figure 1

Intracellular localization of GFP–HAS1 and structure of pericellular hyaluronan coat induced by GFP–HAS1 overexpression. Confocal optical sections of live MCF-7 breast cancer cells transfected with EGFP–HAS1 (green) and stained with fHABC to visualize the hyaluronan coat (red). Localization of EGFP–HAS1 is shown in (A), fHABC in (B), and merged images in (C). Arrows in (A) point patches of signal near the plasma membrane. Scale bar 10 μm. Original data published in Ref. (13).

Figure 2

Comparison of the structure and intensity of the pericellular hyaluronan coat in MCF-7 cells overexpressing the three HAS isoenzymes. Structure of the hyaluronan coat of live MCF-7 cells transfected with fusion proteins Dendra2–HAS1 (A,D), Dendra2–HAS2 (B,E), and Dendra2–HAS3 (C,F) and labeled with fHABC (red). Single confocal sections obtained from the middle level of nucleus (blue) are shown in (A–C). Vertical views created from compressed image stacks of horizontal optical sections are shown in (D–F) to show the dorsal protrusions (arrows). The integrated intensity (mean intensity × area) of hyaluronan coat probed with fHABC in the three HAS transfectants was measured in thresholded area of optical sections through the center of nucleus (G). Mean of three independent experiments is represented (total number of measured cells in each group = 92). Magnification bar in (F), 10 μm. Original data published in Ref. (12, 81).

Figure 3

Glucosamine induces the growth of hyaluronan coat produced by HAS1. Confocal optical sections of pericellular hyaluronan coats on COS-1 cells expressing Dendra2–HAS1 without glucosamine (A–C) and after 6 h incubation with 1 mM glucosamine (D–F). Green, Dendra2–HAS1; red, hyaluronan coat; blue, nuclei. Magnification bars 20 μm. Original data published in Ref. (12, 13).

Figure 4

Subcellular localization of endogenous HAS1 detected by immunostainings. MCF-7 cells transiently transfected with empty vector (A) and HAS1 expressing plasmid (B), followed by immunostaining with polyclonal HAS1 antibodies (brown color). Arrows in (B) show the HAS1 overexpressing cells. A 3D confocal projection of human chondrosarcoma cell (HCS) (C) and transformed mouse embryonic fibroblast (MEF) (D) stained with HAS1 immunofluorescence (green). Arrows in (C,D) point plasma membrane protrusions. Blue, nuclei. Magnification bars in (B,D) = 20 μm. Original data published in Ref. (6, 9, 14).

Figure 5

Localization of HAS1 in breast cancer tissue. A paraffin section of breast carcinoma immunostained with HAS1 polyclonal antibody (brown). Nuclei are labeled blue. A mainly cytoplasmic localization of HAS1 is detected in carcinoma cells (asterisks) and in stromal fibroblasts (arrows). Special accumulation of staining is seen intracellularly (arrowheads). Magnification bar 50 μm. Original data published in Ref. (8).