Expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

Abstract

The expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was analyzed. Western blot analysis showed that three isotypes of caveolins including caveolin-1, -2 and -3 increased significantly in the spinal cords of rats during the early stage of EAE, as compared with the levels in control animals (p < 0.05); the elevated level of each caveolin persisted during the peak and recovery stage of EAE. Immunohistochemistry demonstrated that caveolin-1 and -2 were expressed constitutively in the vascular endothelial cells and ependymal cells of the normal rat spinal cord, whereas caveolin-3 was almost exclusively localized in astrocytes. In EAE lesions, the immunoreactivity of caveolin-1 was increased in the ependymal cells, some astrocytes, and some inflammatory cells of the spinal cord, while that of caveolin-2 showed an intense immunoreactivity. Caveolin-3 was expressed constitutively in some astrocytes, but not in endothelial cells; its immunoreactivity was increased in reactive astrocytes in EAE lesions. The results of the Western blot analysis largely confirmed the observations obtained with immunohistochemistry.

Taking all the findings into consideration, we postulate that the expression levels of each caveolin begin to increase when EAE is initiated, possibly contributing to the modulation of signal transduction pathways in the affected cells.

Introduction

Caveolae are flask-shaped vesicular invaginations of the plasma membrane with diameters of 50–100 nm (Okamoto et al., 1998). They play important roles in a variety of cellular functions, including signal transduction, lipid metabolism, cell growth, and apoptotic cell death (Okamoto et al., 1998). The principal protein components of caveolae are members of the caveolin family of proteins; the genes for three of these proteins, caveolin-1, -2 (Scherer et al., 1996), and -3 (Tang et al., 1996), have been identified to date.

Caveolin-1 has two forms, designated alpha and beta, which share a distinct, but overlapping, cellular distribution and differ by an amino terminal 31-amino-acid sequence that is absent from the beta isoforms (Li et al., 1996). Caveolin-2 was cloned in an effort to identify other novel resident proteins of adipocyte caveolae, where there is an abundance of caveolae and caveolin-1 (Razani et al., 2002). Caveolin-3 was found by cDNA library screening for caveolin-1 homologous genes (Razani et al., 2002). Human caveolin-2 is ∼ 38% identical and 58% similar to human caveolin-1 (Scherer et al., 1996). Caveolin-1 and caveolin-3 are ∼ 65% identical and ∼ 85% similar (Tang et al., 1996). Caveolin-3 is most closely related to caveolin-1 and is most distant from caveolin-2, based on sequence homology (Tang et al., 1996). Functionally, the caveolins might also differ in their interactions with trimeric G-protein α-subunits (Li et al., 1995, Harris et al., 2002).

Although similar in structure and function, these molecules differ in their cellular distribution and specific properties (Harris et al., 2002). Caveolin-1 and -2 are most abundantly expressed in adipocytes, endothelial cells, and smooth muscle cells (Okamoto et al., 1998, Scherer et al., 1997), and are also present in astrocytes (Schlachetzki and Pardridge, 2003, Ikezu et al., 1998), macrophages (Kiss et al., 2002), and Schwann cells (Mikol et al., 2002). The expression of caveolin-3 is thought to be largely muscle-specific (Song et al., 1996), although it has also been detected in astrocytes (Ikezu et al., 1998) and brain microvessels in humans (Virgintino et al., 2002). Caveolin-1 and -2 are up-regulated in response to a number of stimuli and insults, including treatment with nerve growth factor (NGF) and mechanical injury in PC12 cells (Galbiati et al., 1998). As well, the up-regulation of caveolin-3 has been shown to be associated with the processing of amyloid precursor protein in astrocytes in Alzheimer's disease (Nishiyama et al., 1999).

Previous studies indicated that it was highly possible that caveolin-1, -2, and -3 would be involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) because one of the typical pathological findings of EAE is the breakdown of the blood–brain barrier, leading to the infiltration of autoimmune inflammatory cells into the brain and eventually to reactive microgliosis and astrogliosis. EAE is an experimentally induced autoimmune disease of the CNS mediated by CD4+ T cells that is used as an animal model of multiple sclerosis, a human demyelinating disease (Raine, 1994). EAE lesions are characterized by the infiltration of T cells and macrophages into the subarachnoid space during the early stage and the activation of microglia and astrocytes during the peak symptomatic stage of the disease (Shin et al., 1995). Cell activation events, such as increased expression of cell adhesion molecules on vascular endothelial cells (Shin et al., 1995), astrocyte proliferation, activation of brain macrophages, and apoptosis of inflammatory cells, are typical findings in both EAE and multiple sclerosis lesions (Schonrock et al., 1998).

The aim of the present study was to elucidate the expression patterns of caveolin-1, -2, and -3 in the spinal cords of rats with EAE and to confirm the cell phenotypes involved.