The hyaluronan receptor for endocytosis (HARE) activates NF-κB-mediated gene expression in response to 40-400-kDa, but not smaller or larger, hyaluronans

Abstract

The hyaluronan (HA) receptor for endocytosis (HARE; Stabilin-2) binds and clears 14 different ligands, including HA and heparin, via clathrin-mediated endocytosis. HA binding to HARE stimulates ERK1/2 activation (Kyosseva, S. V., Harris, E. N., and Weigel, P. H. (2008) J. Biol. Chem. 283, 15047-15055). To assess a possible HA size dependence for signaling, we tested purified HA fractions of different weight-average molar mass and with narrow size distributions and Select-HA(TM) for stimulation of HARE-mediated gene expression using an NF-κB promoter-driven luciferase reporter system. Human HARE-mediated gene expression was stimulated in a dose-dependent manner with small HA (sHA) >40 kDa and intermediate HA (iHA) <400 kDa. The hyperbolic dose response saturated at 20-50 nM with an apparent K(m) ~10 nM, identical to the Kd for HA-HARE binding. Activation was not detected with oligomeric HA (oHA), sHA <40 kDa, iHA >400 kDa, or large HA (lHA). Similar responses occurred with rat HARE. Activation by sHA-iHA was blocked by excess nonsignaling sHA, iHA, or lHA, deletion of the HA-binding LINK domain, or HA-blocking antibody. Endogenous NF-κB activation also occurred in the absence of luciferase plasmids, as assessed by degradation of IκB-α. ERK1/2 activation was also HA size-dependent. The results show that HA-HARE interactions stimulate NF-κB-activated gene expression and that HARE senses a narrow size range of HA degradation products. We propose a model in which optimal length HA binds multiple HARE proteins to allow cytoplasmic domain interactions that stimulate intracellular signaling. This HARE signaling system during continuous HA clearance could monitor the homeostasis of tissue biomatrix turnover throughout the body.

Keywords: Cell Signaling; ERK1/2; Glycosaminoglycan; Hyaluronate; Scavenger Receptor; Select-HA; Signal Transduction; Stabilin-2; Stress Response; Transcription.

FIGURE 1.

HA size nomenclature based on log incremental mass ranges. Four different 10-fold HA mass ranges are used (top of panel) as follows: oHA (between >1 and 10 kDa); sHA (between >10 and 100 kDa); iHA (between >100 and 1,000 kDa), and lHA (between >1,000 and 10,000 kDa). HA size within any of these four mass ranges is described further by assigning thirtiles (one-thirds) by the descriptors low-, mid-, and high-range. For example (bottom of panel), sizes within the iHA range are further defined as low-range (100–330 kDa), mid-range (330–660 kDa), and high-range (660–999 kDa) iHA. The smallest oHA fragment is actually a tetrasaccharide (technically <1 kDa), and the largest HA size in vivo is unknown but is likely >10 MDa (>lHA). The red dotted lines and spanning double-arrow represent the sHA-iHA region of active HA (∼40–400 kDa) capable of stimulating HARE-mediated signaling and gene activation.

FIGURE 2.

SEC-MALLS and electrophoretic analyses of purified HA preparations. Non-animal-derived, low endotoxin-containing HA preparations were fractionated by SEC and selectively pooled as described under “Experimental Procedures.” A, size distribution for each color-coded HA preparation with the indicated M_w (ranging from 36 to 967 kDa) is plotted as the cumulative weight fraction. The sizes within the vertical dotted lines represent the active HA size range for HARE-mediated stimulation of NF-κB activated gene expression. B, agarose gel electrophoresis of the indicated purified narrow size range HA preparations was performed and the 1.2% gels processed as described under “Experimental Procedures.” For comparison, unfractionated HA preparations (LifeCore) with M_w values of 741 and 215 kDa are shown at the right. The M_w values for Lo- and High-Ladder Select-HA standards in lane M were (kDa) as follows: 30.3, 111, 214, 310, 495, 667, 940, 1138, and 1510.

FIGURE 3.

HA binding to human or rat HARE mediates NF-κB-activated gene expression in a dose-dependent manner. Cells expressing hHARE (A, ●), rHARE (B, ●) or EV (A and B; ○) were grown and transiently transfected with plasmids encoding firefly and Renilla LUC for 18 h in Transfection Medium. Cells were washed, incubated in serum-free medium for 1 h, washed again, and incubated with the indicated concentrations of 107-kDa iHA for 4 h. Cells were then processed and analyzed for their relative ratios of LUC activities as described under “Experimental Procedures.” Results are normalized to the untreated control and expressed as a fold-change in the ratio of firefly-to-Renilla LUC activity. In Figs. 3–9, values are means ± S.E. (n = 9) from three independent experiments, unless noted otherwise. Values for p compare HARE and EV cells at each HA concentration and HARE cells plus HA versus EV cells without HA. Only sample sets with significant differences in both cases are marked: ***, p < 0.001; ****, p < 0.0001.

FIGURE 4.

HA-HARE binding is required for NF-κB-activated gene expression. A, cells expressing hHARE (black bars), hHARE(ΔLink) (gray bars), or EV (white bars) were incubated with nothing or 50 nm 107-kDa HA and processed as in Fig. 3 (****, p < 0.0001; n = 9). B, cells expressing rHARE were incubated with 50 nm 107-kDa HA, 30 μg/ml mAb-174 mAb, or mouse IgG alone for 4 h and/or with HA added after preincubation with mAb-174 for 30 min and then processed as in Fig. 3 (*, p < 0.05; n = 9).

FIGURE 5.

HA size dependence for HARE-mediated NF-κB-activated gene expression. EV (white bars) or hHARE (black bars) cells were incubated with 20 nm (A) or 100 nm (B) preparations of narrow size range HA of the indicated M_w values, representing sizes from the oHA-sHA boundary to high-range iHA, for 4 h, and then processed as in Fig. 3. C, dose-response curves expressed in weight concentration units (μg/ml) rather than molar units (nm) are shown using active 107 kDa and inactive 14- or 509-kDa HA preparations. Values for p comparing hHARE and EV cells (n = 9) are as follows: *, p < 0.05; **, p < 0.005; ***, p < 0.001; ****, p < 0.0001.

FIGURE 6.

Select-HA size dependence for HARE-mediated NF-κB-activated gene expression. EV (white bars) or hHARE (black bars) cells were incubated with 100 nm of the indicated nearly monodisperse Select-HA for 4 h and processed as in Fig. 3. Values for p compare cell responses at each HA concentration (n = 9): **, p < 0.005; ***, p < 0.001; ****, p < 0.0001.

FIGURE 7.

Rat and human HARE show similar HA size dependence for NF-κB-activated gene expression. EV (white bars), hHARE (black bars), or rHARE (gray bars) cells were incubated with 20 nm of different narrow size range HA preparations with the indicated M_w values for 4 h and processed as in Fig. 3. Values for p compare hHARE or rHARE with EV cells for each HA sample (n = 6): *, p < 0.05.

FIGURE 8.

Low-range iHA stimulation of HARE-mediated NF-κB activation is blocked by smaller or larger HA. A, EV (white bars) and hHARE (black bars) cells were incubated with 137-kDa low range Select-iHA and the indicated concentrations of 509-kDa mid-range iHA for 4 h and processed as in Fig. 3. Values for p compared hHARE and EV cells at each HA concentration (n = 9) are as follows: **, p < 0.005; ****, p < 0.0001. B, hHARE cells were incubated with 137-kDa low-range Select-iHA and the indicated concentrations of narrow range 14-kDa oHA-iHA. p values compared 10 nm 137-kDa HA samples without versus with 14-kDa HA (*, p < 0.05; for 0 versus 100 nm: n = 9).

FIGURE 9.

HARE-mediated NF-κB activation by polydisperse HA occurs only if the active HA size fraction is high enough. A, cells expressing hHARE were incubated with medium alone (no addition) or 20 nm of polydisperse 51 or 741 kDa or 40 nm of a 1:1 mixture and processed as in Fig. 3. Significant differences are indicated by a symbol above a bar (left of line) for comparison with the no addition sample or by a symbol to the right of the line for comparison with the 51-kDa sample (*, p < 0.05; ****, p < 0.0001; n = 9). SEC-MALLS analysis of the cumulative weight fractions of 51-kDa (B) and 741-kDa (C) HA reveals that the fraction of active HA in the 40–400-kDa range (unshaded area) is greater than the inactive fraction (gray shaded area) in the 51-kDa but not in the 741-kDa HA preparations.

FIGURE 10.

Effect of TNF-α and HA on IκB-α degradation. EV (A, C, E, and G) or hHARE (B, D, F, and H) cells were grown to confluence and washed. After a 1-h serum-free medium incubation at 37 °C, the cells were incubated with 1 ng/ml TNF-α (A, B, E, and F) or 100 nm 137-kDa HA (C, D, G, and H) for 0–180 min as indicated. Cells were processed and Western blot analyses performed with a mAb against IκB-α (A–D, top of panels) as under “Experimental Procedures.” The same membranes were stripped and reprobed with anti-actin Ab as an internal loading control (A–D, bottom of panels). Blots from three independent experiments were digitized by scanning, and densitometric analysis was performed to determine the IκB-α/α-actin ratios at each time. Normalized data (E–H) are presented as mean ± S.E. (n = 3) percent of the IκB-α/α-actin ratio relative to the no addition time 0 value as 100% (E–H); *, p < 0.05.

FIGURE 11.

HARE-mediated ERK activation also shows HA size dependence. EV (A and C) or hHARE (B and D) cells were grown to confluence, washed, incubated in serum-free medium at 37 °C for 1 h, and then incubated with or without 10 μg/ml 80-kDa (A and B) or 560-kDa (C and D) HA for the indicated times. Lysate samples were subjected to 10% SDS-PAGE and Western analysis with Ab against phospho-ERK1/2 (pERK1/2) and then, after stripping, with Ab against total ERK1/2 protein (tERK1/2) and anti-actin (31). Blots from three to four independent experiments were digitized, and densitometric analysis was performed to determine the phospho-ERK/total ERK ratios at each time. Values are the mean ± S.E. (n = 3–4) percent of the phospho-ERK/total ERK ratio relative to time 0 (the no addition value) as 100%. p values compare the sample pairs at time 0 and the indicated time (*, p < 0.05; **, p < 0.005; ***, p < 0.001).

FIGURE 12.

Model for the HA size dependence of HARE-mediated cell signaling. The scheme shows several possibilities for how two HARE proteins are able to interact with and bind to the same HA molecule, depending on the mass, and thus length, of the HA. Signaling does not occur with oHA or sHA <40 kDa because HA of this size is only able to bind one HARE protein (left). Signaling HA, between 40 and 400 kDa, is long enough to bind to two HARE molecules and yet short enough that the two proteins are brought into close proximity, inducing their cytoplasmic domains to interact and create complexes with signaling molecules (middle). Two proteins are shown, but three, four, or more HARE molecules could interact in a similar HA size-dependent manner to achieve cytoplasmic domain signaling complexes (e.g. trimers or tetramers). Complexes could occur in which two or more HARE proteins bind with the same HA to create dimers, dimers of dimers (tetramers), or a larger closed circular complex in which >4 cytoplasmic domains are brought together. As HA length increases, the bound HARE proteins are more likely to be further apart and not interact (right), even though more than two receptors may bind to the same HA; monomeric HA-HARE complexes may also occur.