A Requirement for the CD44 Cytoplasmic Domain for Hyaluronan Binding, Pericellular Matrix Assembly, and Receptor-Mediated Endocytosis in COS-7 Cells

CD44-negative COS-7 cells were transfected with expression constructs for CD44H (the predominant CD44 isoform), CD44E (epithelial isoform), or truncation mutant derivatives lacking the carboxyl-terminal 67 amino acids of the cytoplasmic domain, CD44HDelta67 and CD44EDelta67. The truncation mutant CD44HDelta67 is identical to a naturally occurring alternatively spliced "short tail" CD44 isoform (CD44st), which incorporates exon 19 in place of exon 20. CD44st lacks intracellular signaling motifs as well as protein domains necessary for interaction with cytoskeletal components. Transfection of COS-7 cells with each construct yielded equivalent levels of mRNA expression, whereas no CD44 expression was observed in parental, nontransfected COS-7 cells. Western analysis and immunostaining of COS-7 transfectants confirmed CD44 protein expression of the truncation mutant derivatives. COS-7 cells transfected with CD44H or CD44E gained the capacity to bind fluorescein-conjugated HA (fl-HA) and assemble HA-dependent pericellular matrices in the presence of exogenously added HA and proteoglycan. In addition, the CD44H- and CD44E-transfected cells were able to internalize surface-bound fl-HA. COS-7 cells transfected with the vector alone or with either of the mutant CD44 isoforms, CD44HDelta67 or CD44EDelta67, did not exhibit the capacity to assemble pericellular matrices or to bind and internalize the fl-HA. Cotransfection of CD44Delta67 mutants together with CD44H reduced the size of the HA-dependent pericellular matrices. Transfection of bovine articular chondrocytes with CD44Delta67 also inhibited pericellular matrix assembly. Collectively, these results indicate an obligatory requirement for the CD44 receptor cytoplasmic domain for ligand (HA) binding, formation and retention of the pericellular matrix, as well as CD44-mediated endocytosis of HA. In addition, the results suggest a potential regulatory role for the differentially expressed alternatively spliced short tail CD44 isoform.

having a cytoplasmic tail domain containing only 3 amino acids (encoded by exon 18) and, consequently, lacks intracellular signaling motifs (14,15) as well as protein domains necessary for interaction with cytoskeletal components. Currently, the biological function of the CD44st isoform is unknown leading one to query whether the expression of this unique isoform is of physiological significance. However, we have shown that IL-1α induces in a sharp, dosedependent increase in CD44st mRNA in chondrocytes resulting in an increase in the proportion of this isoform relative to CD44H (13). This has led us to hypothesize that CD44st exerts potential modulatory effects on CD44H-mediated functions such as would be observed by a dominant-negative isoform.
In an effort to gain a better understanding of the specific functions of the CD44st isoform expressed by chondrocytes, CD44-negative COS-7 cells were transfected with expression constructs containing truncation mutant derivatives of human CD44 that, like the naturallyoccuring CD44st, lack the carboxyl terminal 67 amino acids of the cytoplasmic domain. The truncation mutants were prepared by point mutation of human CD44H as well as CD44E.
CD44E represent the so-called "epithelial" isoform of CD44 containing an extended extracellular protein domain due to translation of three additional alternative spliced, exons 12-14, within the extracellular domain. Articular chondrocytes were also transfected with the CD44 mutant isoform. We present results that demonstrate a mandatory requirement for the CD44 receptor co-transfectants of pCD44H and pCD44E∆67 were prepared using 2.0 µg/ml of each plasmid. In order to identify transfected cells, some of the cDNA constructs namely, CD44H and CD44E were subcloned into a green fluorescence protein (GFP) expressing vector (pTracer-SV40).
Samples were standardized to 10 µg protein and loaded onto 8% SDS-polyacrylamide gels for electrophoresis. Following electroblot transfer onto nitrocelluose, CD44H, CD44H∆67, CD44E and CD44E∆67 were detected following a 2 h incubation with anti-human CD44 monoclonal antibody (BU-52) followed by alkaline phosphatase conjugated anti-mouse IgG and enhanced chemiluminescence substrate (diethanolamine) substrate. The resultant bands were detected using a fluoroimaging system (Storm FluorImager 860, Molecular Dynamics, Sunnyvale, CA). Visualization of Matrix Assembly --Cell-associated pericellular matrices were visualized using a particle exclusion assay (2). Endogenous matrix on bovine articular chondrocytes ( Binding and Uptake of Exogenous HA by Transfected COS-7 Cells--COS-7 cells were cotransfected with various CD44 constructs in pCDM8 together with a RFP-positive empty vector.

Construction of CD44 mock short-tail mutants pCD44H∆67 and pCD44E∆67--Full-length
CD44H and CD44E cDNAs were subcloned as Hind III-Not I fragments from pπH3H-CD44H or pπH3H-CD44E (16) into the expression vector pCDM8. The CD44 cytoplasmic domain "mock short-tail" expression mutants, pCD44H∆67 and pCD44E∆67, were created by the introduction To assess if the various CD44 constructs were being appropriately expressed as CD44 protein, total cell lysates were extracted from COS-7 cells for each transfection group.
Equivalent protein aliquots (10 µg) were subjected to electrophoresis on an 8% SDSpolyacrylamide gel. Western blot analysis was performed using an anti-human CD44 monoclonal antibody that recognizes an epitope contained within the receptor extracellular domain, present in all of the CD44 expression constructs ( inserted DNA sequence (Fig. 4E). In addition, use of control isotype-specific control IgG or, pretrypsinization of these cells prior to incubation with BU-52 also resulted in a negative cell reaction (data not shown). Thus, translation of the carboxy-terminal truncated mutant CD44 proteins did not result in their enhanced degradation intracellularly or a failed translocation of these modified proteins to the plasma membrane.

Pericellular matrix assembly on live COS-7 cells transfected with CD44 expression constructs--
In an effort to determine the requirement of the cytoplasmic domain of CD44 in pericellular matrix assembly, an important chondrocyte function, transfected COS-7 cultures were supplemented with exogenous, purified HA and cartilage-derived aggrecan proteoglycan.
Previous studies from our laboratory demonstrated that following successful transfection with pCD44H or pCD44E cDNA, COS-7 cells gained the capacity to capture and assemble pericellular matrices in the presence of these two matrix macromolecules (16). Consistent with our previous findings, neither untreated COS-7 cells, nor COS-7 cells transfected with control pCDM8 vector had the capacity to assemble pericellular matrices ( in order for COS-7 cells to gain a capacity to bind and internalize HA, receptors such as CD44H or CD44E must be expressed. In the COS-7 cells transfected with pCD44H∆67 (RFP-positive cells) little staining for cell surface-bound fl-HA was observed (Fig. 6F). However in a few cells, a thin but still discernable layer of fl-HA binding to the cell surface could be seen (Fig. 6G). This suggests that the mock short tail isoforms of CD44 still exhibit at least a limited capacity for HA binding. Nonetheless, no intracellular vesicular clusters of fl-HA were evident in pCD44H∆67 transfectants subsequent to trypsin treatment (Fig. 6H). Like the pCD44H∆67 transfectants, the pCD44E∆67 also failed to display internalized fl-HA (data not shown). Thus, the presence of an intracellular tail is likely an absolute requirement for efficient HA endocytosis either because its absence limits HA binding at the cell surface and/or, its absence prevents interactions with cytosolic components necessary for the internalization events.   Fig. 7, increasing the proportion of CD44H∆67 expression resulted in a proportional decrease in the number of cells with large pericellular matrices. The proportion of cells with morphometric ratios less than 1.5 increased from 39.5% (pCD44H and no pCD44H∆67) to 71.3% when the ratio of pCD44H∆67 to pCD44H was 1:1. Similar to the results depicted in Fig. 5, 98.2% of the COS-7 cells transfected with pCD44H∆67 alone had morphometric ratios less than 1.5 (Table 1). Thus, the expression of the short tail isoform not only results in the appearance of a non-ligand-binding receptor but also, its presence perturbs the function of long tail CD44 even when sufficient levels of long tail receptor are present.

Transfection of bovine articular chondrocytes with CD44H∆67 and pericellular matrix
assembly-The data shown in Figure 7 suggested that the expression of CD44H∆67 inhibited the functions of the wild type CD44. To investigate this is a more physiological setting, primary cultures of bovine articular chondrocytes were transfected with pCD44H∆67. Using FuGENE reagent and pre-incubation with Streptomyces hyaluronidase, transfection efficiencies in the range of 10-30% were obtained. As with the COS-7 cells, the expression of GFP allowed the identification of successfully transfected cells. Figure  Thus the loss of pericellular matrices observed in Figure 8A was not due to a transfection-related toxicity (e.g., GFP expression) or mere over-expression of a recombinant membrane protein.

DISCUSSION
The major findings of this study are that short tail CD44 isoforms (CD44H∆67 and CD44E∆67) do not support binding of HA to the plasma membrane, the assembly of pericellular matrices and consequently, do not mediate the internalization of bound HA. In addition, the over-expression of the short tail CD44 isoform together with long tail CD44 results in the inhibition CD44-mediated matrix assembly. Besides experimental applications of such results there is physiological significance as a protein identical to CD44H∆67 is expressed in chondrocytes due to alternative splicing to generate exon 19-containing CD44.
Human articular chondrocytes naturally express both exon-19 and exon-20 containing CD44 mRNA transcripts. The proportion of exon-19 to exon-20 mRNA varies from donor to donor but typically falls within the range of 15% to 33% of the total CD44 message (13). This proportion can also be observed at the level of CD44 protein (13). We are not the first to document the expression of exon-19 containing CD44 mRNA. For example, short tail isoform mRNAs were detected in several lymphoid as well as nonlymphoid cells such as foreskin fibroblasts (18).
These investigators also described the expression of CD44E long tail and CD44E tail-less transcripts in a keratinocyte cell line (18). However, the level of short tail expression in these cell types was reported as 0.5 to 1.0% of the total CD44 mRNA, much lower than what we observe in chondrocytes. Given that a naturally-occurring short tail CD44 is expressed by chondrocytes, and that the levels of expression vary considerably, we sought to determine the physiological role for the short tail CD44 isoform. Does short tail CD44 exhibit functional properties that are identical to long tail CD44, distinct from the long tail isoform or, does its expression in some way act to modulate functions attributed to full length CD44?
One CD44 function that has received considerable attention is the regulation of HA binding. minimum requirement. One conclusion that can be drawn from these observations is that the role of the short tail CD44 isoform cannot be as a "decoy" receptor because it has little native affinity to bind its ligand and unlikely competes with wild-type CD44 for HA.
Another suggested function of CD44 is to mediate the endocytosis of HA. One of the highlights of this study was that COS-7 cells transfected with either pCD44H or pCD44E gained the capacity to internalize bound HA. Although the involvement of CD44 has been implicated in several studies (29)(30)(31)(32)(33), including our own (8,4,34), to our knowledge the participation of CD44 in HA endocytosis has never been heretofore directly demonstrated by a "gain-of-function" approach. This implies that the monkey kidney epithelial cells (          Morphormetric analysis of changes in pericellular matrix size present on COS-7 cells transfected with CD44 constructs as described in Fig. 7.