The Hyaluronan Receptor for Endocytosis Mediates Hyaluronan-dependent Signal Transduction via Extracellular Signal-regulated Kinases

Abstract

The hyaluronan (HA) receptor for endocytosis (HARE) mediates the endocytotic clearance of HA and other glycosaminoglycans from lymph and blood. Two isoforms of human HARE, 315-and 190-kDa, are highly expressed in sinusoidal endothelial cells of liver, lymph node, and spleen; HARE is also in specialized cells in the eye, heart, brain, and kidney. Here we determined whether HA binding to HARE initiates intracellular signaling in Flp-In 293 cells stably expressing either the 315-and 190-kDa HARE or the 190-kDa HARE alone. HARE was co-immunoprecipitated with extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 members of the mitogen-activated protein kinase signaling cascade. ERK phosphorylation increased in a dose-and time-dependent manner when HA was added to cells expressing full-length or 190-kDa HARE, but not cells with vector-only or a HARE(⌬Link) construct with greatly decreased (ϳ90%) HA uptake. HA did not induce phosphorylation of JNK or p38. A maximum increase in phospho-ERK1/2 occurred within 30 min at 5 g/ml HA, and the response was dampened at >20 g/ml HA. HA binding did not increase the level of HARE-ERK complexes, but did increase HARE phosphorylation. These findings demonstrate a novel functional response, when HARE binds HA, that leads to activation of ERK1/2, important mediators of intracellular signal transduction. HA plays important roles in matrix assembly, cell differentiation, migration, morphogenesis, and wound healing (1-3). Elevated levels of HA are associated with various pathologies, such as arthritis, inflammation, and cancer (4 -7). The average adult human contains ϳ15 g of HA, of which ϳ5 g is synthesized and degraded daily in tissues throughout the body (1). Most of the HA and CS types released from tissues during this turnover process are ultimately cleared from the circulation and lymph fluid by the HA Receptor for Endocytosis (HARE) (8, 9), also known as Stabilin-2 (10) or FEEL-2 (11). Human HARE is encoded by the 180-kb STAB2 gene, found on chromosome 12, consisting of 69 exons, and is abundantly expressed in the sinusoidal cells of lymph nodes, liver, and spleen (8 -10, 12, 13). Rat and hHARE in these tissues are present as two isoforms (8, 12, 13), e.g. hHARE isoforms are ϳ190 and ϳ315 kDa. Although HARE in the sinusoidal cells of liver and lymph node has a known endocytic clearance function, it may also have other, not yet described, functions. HARE is also expressed in corneal and lens epithelium, in mesenchymal cells of heart valves, in ependymal cells lining the ventricles in the brain, in epithelial cells covering the renal papillae (14), and in oviduct (15). The functions of HARE in these latter tissues are unknown and might be different from local GAG clearance.We have stably expressed the recombinant 190-kDa (16) and full-length 315-kDa (17) HARE proteins in Flp-In 293 cell lines, using cDNA derived from human lymph node. These Flp-In cell lines have one unique, recombinase-mediated integration site. The full-length 315-k...

Hyaluronan Anchored to Activated CD44 on Central Nervous System Vascular Endothelial Cells Promotes Lymphocyte Extravasation in Experimental Autoimmune Encephalomyelitis

“…Interestingly, antibody blockage of HA binding to HA receptor for endocytosis impairs tumor cell metastasis, a process likened to active lymphocyte extravasation (37,38). HA receptor for endocytosis is also capable of initiating intracellular signaling via MAP kinases (39). If a redundant or compensatory mechanism for HA binding on activated lymphocytes exists, it may function beyond the initial capture and rolling of lymphocytes to influence cellular rearrangement in preparation for extravasation.…”

The Hyaluronan Receptor for Endocytosis (HARE) Activates NF-κB-mediated Gene Expression in Response to 40–400-kDa, but Not Smaller or Larger, Hyaluronans

“…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 g/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.…”

“…11). Cell extracts were processed for Western analyses to detect ERK1/2 activation as described previously (31). As expected, neither HA size had any effect on ERK1/2 activation in EV cells (Fig.…”

“…Adult humans contain ϳ15 g of tissue HA and synthesize and degrade one-third of this amount daily. Native lHA is continuously partially degraded by an unknown mechanism and released from tissue ECMs as ϳ1-MDa fragments that may contain bound proteins such as growth factors and lecticans with CS and other glycosaminoglycan chains (31)(32)(33). These HA-proteoglycan fragments and associated components enter the lymphatics and lymph nodes, the initial and primary sites for 85% of the HA and CS clearance and degradation.…”

“…This HARE receptor signaling system in response to HA clearance could play a role in monitoring the status of tissue biomatrix turnover throughout the body. (25,31) using Flp-In 293 (HEK) cells, which are engineered to contain a selected recombinase insertion site, and the correct single insertion was verified. The Invitrogen protocol was followed to confirm that the clones selected contained one transgene inserted at the correct locus.…”