The deubiquitinating enzymes USP4 and USP17 target hyaluronan synthase 2 and differentially affect its function

Abstract

The levels of hyaluronan, a ubiquitous glycosaminoglycan prominent in the extracellular matrix, is balanced through the actions of hyaluronan-synthesizing enzymes (HAS1, 2 and 3) and degrading hyaluronidases (Hyal 1, 2, 3 and PH20). Hyaluronan accumulates in rapidly remodeling tissues, such as breast cancer, due to deregulated expression of the HAS2 gene and/or alterations of HAS2 activity. The activity of HAS2 is regulated by post-translational modifications, including ubiquitination. In order to identify deubiquitinating enzymes (DUBs) that are involved in de-ubiquitination of HAS2, a complementary (cDNA) library of 69 Flag-HA-tagged human DUBs cloned into retroviral vectors was screened in human embryonic kidney (HEK) 293T cells for their ability to de-ubiquitinate myc-tagged HAS2. Several DUBs were found to decrease the ubiquitination of 6myc-HAS2, among which, the most effective were USP17 and USP4. USP17 efficiently removed polyubiquitination, whereas USP4 preferentially removed monoubiquitination of 6myc-HAS2. Co-immunoprecipitation studies revealed interactions between HAS2 and USP17, as well as between HAS2 and USP4, in membrane preparations of HEK293T cells. USP17 significantly stabilized 6myc-HAS2 protein levels, whereas USP4 did not. The silencing of USP17 led to decreased hyaluronan production, whereas the suppression of USP4 increased hyaluronan synthesis. Importantly, high levels of USP17 and HAS2 were detected in a panel of cancer cell lines compared to normal cells, and immunohistochemical stainings revealed higher expression of USP17 and HAS2 in tissues of lung cancer patients compared to normal tissue. In conclusion, USP17 and USP4 differently affect HAS2 ubiquitination, and the stability and function of HAS2.

TRAF4/6 Is Needed for CD44 Cleavage and Migration via RAC1 Activation

“…Human lung adenocarcinoma A549 cells (kindly provided by A. Moustakas, Uppsala University, purchased from American Type Culture Collection (ATCC)), non-small-cell lung carcinoma H1299 cells [ 56 ], glioblastoma U251MG cells [ 57 ], and prostate cancer PC3U cells [ 23 ] were grown in RPMI-1640 (Sigma-Aldrich Sweden AB, Stockholm, Sweden), whereas wild-type (Wt) or Traf6-deficient ( Traf6 -/- ) mouse embryonic fibroblasts [ 23 ], human breast cancer Hs578 cells [ 56 ], and a bone-metastasizing clone of human breast cancer MDA MB 231 cells [ 58 ] were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich Sweden AB, Stockholm, Sweden), all in the presence of 10% fetal bovine serum (FBS) (Biowest, Biotech-IgG AB, Lund, Sweden), penicillin and streptomycin (100 X/mL), and 5 mM L-glutamine (Sigma-Aldrich Sweden AB, Stockholm, Sweden). Human colon cancer HCT-116 and CACO-2 cells were grown in McCoy′s 5a medium (Sigma-Aldrich Sweden AB, Stockholm, Sweden) and in Minimum Essential Medium Eagle (Sigma-Aldrich Sweden AB, Stockholm, Sweden), respectively (both cell lines were kindly provided by Dr I. Ferby, Uppsala University, being purchased from ATCC).…”

Activated hyaluronan metabolism in the tumor matrix — Causes and consequences

“…First, O-GlcNAc moieties coupled to certain serine/threonine residues strongly increase the lifetime of HAS2 and HAS3 in cultured cells [75,76]. Ubiquitination is another post-transcriptional signaling system that contributes to HAS2 enzyme activity and lifetime [77,78]. HAS2 enzymes are also phosphorylated, the effect on function depending on the site of the phosphorylation [79][80][81].…”

Tricho-rhino-phalangeal syndrome 1 protein functions as a scaffold required for ubiquitin-specific protease 4-directed histone deacetylase 2 de-ubiquitination and tumor growth

“…The ubiquitin-specific peptidase 4 (USP4) is proposed to be a potential oncogene, which can transform NIH3T3 cells [ 19 ], and USP4-deficient murine embryonic fibroblasts exhibit retarded growth [ 20 ]. Previous studies indicate that, compared to normal cells, USP4 is overexpressed in malignant cells [ 21 ]. Recently, USP4 was reported to de-ubiquitinate and stabilize HDAC2, which then inhibits p53 and NF-kB [ 22 ].…”