The intracellular proteolytic fragment of CD44 alters CD44 function in chondrocytes

Abstract

CD44 is an adhesion molecule involved in several biological functions and the primary receptor for hyaluronan (HA). In cartilage, CD44 participates in the interplay between cells and the extracellular matrix to fine tune cellular responses to cytokines and growth factors. CD44 thus can act as a mediator between individual chondrocytes and the extracellular matrix. The extracellular domain of CD44 binds to the HA/proteoglycan rich extracellular matrix, while the intracellular domain interacts with receptors like Smad1 and with the actin cytoskeleton inside the cell. Recently, we have demonstrated that chondrocyte CD44 is proteolytically cleaved from the cell surface by an endogenous, sequential, two step process. First, there is an initial extracellular cleavage of CD44 by a matrix metalloproteinase, which releases a soluble CD44 in the extracellular matrix, followed by a gamma-secretase transmembrane cleavage, which releases an intracellular tail domain of CD44. The main focus of this study is to understand the effects of releasing a CD44 intracellular domain (ICD) that occurs via the action of the gamma-secretase cleavage, and how intracellular accumulation of such fragments might interfere with endogenous, intact, CD44 function. The first aim evaluates whether intracellular accumulation of ICD fragments can affect CD44 ability to bind to HA and retain a pericellular coat by acting as a negative competitor. Another aim was to explore the mechanisms responsible for any effects ICD over-expression might have on CD44 function. Our data suggests that intracellular CD44-ICD over-expression exerts dominant-negative effects on full length native CD44 by competing with binding to the cytoskeleton via ankyrin-3. This is the first study that looks at possible effects of releasing multiple ICD fragments via gamma-secretase cleavage in chondrocytes and as such, will provide useful information on possible significance of CD44 fragmentation in cartilage during osteoarthritis.