KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization

Abstract

Hyaluronan (HA) has an extraordinarily high turnover in physiological tissues, and HA degradation is accelerated in inflammatory and neoplastic diseases. CD44 (a cell surface receptor) and two hyaluronidases (HYAL1 and HYAL2) are thought to be responsible for HA binding and degradation; however, the role of these molecules in HA catabolism remains controversial. Here we show that KIAA1199, a deafness gene of unknown function, plays a central role in HA binding and depolymerization that is independent of CD44 and HYAL enzymes. The specific binding of KIAA1199 to HA was demonstrated in glycosaminoglycan-binding assays. We found that knockdown of KIAA1199 abolished HA degradation by human skin fibroblasts and that transfection of KIAA1199 cDNA into cells conferred the ability to catabolize HA in an endo-β-N-acetylglucosaminidase-dependent manner via the clathrin-coated pit pathway. Enhanced degradation of HA in synovial fibroblasts from patients with osteoarthritis or rheumatoid arthritis was correlated with increased levels of KIAA1199 expression and was abrogated by knockdown of KIAA1199. The level of KIAA1199 expression in uninflamed synovium was less than in osteoarthritic or rheumatoid synovium. These data suggest that KIAA1199 is a unique hyaladherin with a key role in HA catabolism in the dermis of the skin and arthritic synovium.

Fig. 1.

HA degradation via KIAA1199 and regulation of KIAA1199 expression by histamine and TGF-β1 in human skin fibroblasts. (A) Detroit 551 skin fibroblasts were cultured with [³H]HA for 48 h and HA fragments in the culture medium were examined by size-exclusion chromatography. (B) Expression of CD44, HYAL2, and HYAL1 by RT-PCR. GAPDH, a loading control. (C and D) CD44 and HYAL2 were knocked down by treating cells with siRNAs to CD44 or HYAL2. For controls, the cells were transfected with control nonsilencing siRNA (Control siRNA). Cells with siRNA were cultured with [³H]HA for 48 h, and degraded HA was examined by size-exclusion chromatography. Knockdown efficiency for CD44 and HYAL2 was evaluated by immunoblotting (Insets). GAPDH, a loading control. Representative data for two siRNAs are shown. (E) Effect of histamine and TGF-β1 on HA depolymerization. Cells were treated with or without histamine or TGF-β1 and cultured with [³H]HA for 48 h. The HA-degrading activity was analyzed by chromatography. Control, untreated cells. (F) Abrogation of HA-degrading activity by knockdown of KIAA1199 with two different siRNAs specific for KIAA1199. The cells were cultured with [³H]HA for 48 h, and HA degradation was determined. (Inset) Immunoblotting for KIAA1199 and GAPDH (a loading control). (G) Kinetic study of HA degradation by cells treated with or without histamine for 72 h. Control and Histamine, cells treated with vehicle alone and histamine, respectively. (H and I) The expression levels of KIAA1199 mRNA and protein in cells treated with histamine, TGF-β1 or vehicle alone (Cont) for 24 h. Levels of mRNA and protein expression were measured by real-time PCR and immunoblotting. Values (relative mRNA expression, fold KIAA1199 to GAPDH) represent mean ± SD (n = 3). The Dunnett test was used for statistical analysis. The protein expression levels (ratio of KIAA1199 to GAPDH) were estimated by using densitometric scanning Multi Gauge v.2.1 (Fuji Film). A representative finding of three different experiments is shown.

Fig. 2.

Expression of KIAA1199 by dermal fibroblasts in normal human skin. (A) The mRNA expression of KIAA1199 was examined by in situ hybridization using antisense (Left) and sense (Right) RNA probes. (B) KIAA1199 protein expression was analyzed by immunohistochemistry with anti-KIAA1199 Ab (Left) and control rat IgG_2aκ (Right). (Insets) High-power views of the boxed areas. Arrows, KIAA1199⁺ cells. (Scale bars, 50 μm; Insets, 20 μm.) Representative data from three subjects are shown.

Fig. 3.

HA depolymerization by KIAA1199 transfectants and determination of HA cleavage sites. (A and B) HEK293 and COS-7 cells were transiently transfected with empty vector (Mock) or vector containing KIAA1199 cDNA, and then incubated with [³H]HA for 24 h. HA depolymerization was examined by size-exclusion chromatography. Expression of KIAA1199 protein in Mock and KIAA1199 transfectants was assessed by immunoblotting (Insets). (C and D) Determination of the reducing and nonreducing terminal sugars of depolymerized HA. HPLC pattern of pyridylaminated N-acetylglucosamine (PA-GLcNA) obtained from HA depolymerized by KIAA1199/HEK293 cells (C). Sephadex G-25 column chromatogram of depolymerized [³H]HA after incubation with β-N-acetylglucosaminidase (○) or β-glucuronidase followed by incubation with β-N-acetylglucosaminidase (●) (D).

Fig. 4.

Clathrin-specific HA depolymerization and HA-specific binding of KIAA1199 in KIAA1199/HEK293 cells. (A–C) CHC (A), α-adaptin (B), and caveolin-1 (C) were knocked down by siRNAs to each gene in KIAA1199/HEK293 cells. For controls, cells were transfected with control nonsilencing siRNA. The cells were incubated with [³H]HA for 6 h, and HA fragments in the media were analyzed by size-exclusion chromatography. Efficiency of the knockdown was evaluated by immunoblotting (Insets). Representative data from two siRNAs are shown. Note that knockdown of CHC and α-adaptin but not caveolin-1 decreased HA depolymerization. (D) Coimmunoprecipitation of CHC with KIAA1199. Cell lysates were immunoprecipitated with control IgG2aκ or anti-KIAA1199 antibody, followed by immunoblotting (IMB) for KIAA1199 and CHC. Input is shown in the Upper panel. IP, immunoprecipitation. (E) Effects of inhibitors on HA depolymerization. KIAA1199/HEK293 cells were incubated for 3 h in the absence or presence of monensin, NH₄Cl, bafilomycin A1, dynasore, or nocodazol, followed by additional incubation for 6 h with [³H]HA. HA depolymerization was determined by chromatography. (F) GAG-binding assay for KIAA1199 protein. Cell lysates of KIAA1199/HEK293 cells were incubated with H₂O (negative control) or unlabeled HA (HA-H2), chondroitin sulfate A, C, and D (CSA, CSC, and CSD), dermatan sulfate (DS), heparin (Hep), and heparan sulfate (HS) (Upper), or HA-H2, HA-M2, HA-L2, HA-S2 or HA-T2 (Lower). The samples were precipitated with cetylpyridium chloride and analyzed by NuPAGE and immunoblotting with anti-KIAA1199 antibody. (G) KIAA1199 binding to HA-Sepharose. Cell lysates were incubated with control or HA-coupled Sepharose 4B beads (Upper), and were preincubated with H₂O (control) or GAGs before application to HA-Sepharose (Lower). Bound materials to the beads were eluted with NuPAGE LDS sample-loading buffer, and analyzed by immunoblotting with anti-KIAA1199 antibody.

Fig. 5.

KIAA1199-mediated HA degradation in OA and RA synovial fibroblasts, and expression of KIAA1199 by synovial lining and sublining cells in the RA patients. (A) Synovial fibroblasts from a normal subject (n = 1), OA, and RA patients (n = 3 each) were treated with control nonsilencing siRNA or siRNAs for KIAA1199, and incubated with [³H]HA for 48 h. HA depolymerization was analyzed by size-exclusion chromatography. Protein expression of KIAA1199 was assessed by immunoblotting (Insets). Representative data of three OA (OA-2) and RA synovial fibroblasts (RA-3) are shown, and data of other patients are presented in Fig. S7. Representative data from two siRNAs are shown. (B) The expression of KIAA1199 mRNA (Upper) and protein (Lower) in cultured normal (n = 1), OA (n = 3), and RA (n = 3) synovial fibroblasts was analyzed by real-time PCR and immunoblotting. GAPDH, loading control. (C) The expression levels of KIAA1199 in synovial tissues from the patients with noninflammatory joint disease (n = 3), OA (n = 10) or RA (n = 8) were determined by real-time PCR. Data are presented as a scatter blot with mean ± SD. Statistical analysis was done by the Dunnett test. (D and E) Identification of cells expressing KIAA1199 in RA synovial tissue. The expression of KIAA1199 mRNA and protein was determined by in situ hybridization using antisense (Left) and sense (Right) RNA probes (D) and by immunohistochemistry with anti-KIAA1199 Ab (Left) and control rat IgG_2aκ (Right) (E). Arrows, KIAA1199-expressing cells. (Scale bars, 50 μm.) Representative data of eight subjects are shown.