Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer (∼150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.Multiple sclerosis (MS) is a debilitating white matter disease of the central nervous system (CNS). Although much of the evidence from animal models and MS suggests it to be an autoimmune disorder mediated by TH-1 type T cells,¹ other possible causes include genetic and environmental factors, antibody-dependent cytotoxicity, and bacterial and viral infections that may mediate altered protein expression resulting in inflammation, axonal and oligodendrocyte damage, demyelination and CNS scarring.² Growth and survival factors that protect against axonal and oligodendrocyte damage or loss, and dampen the inflammatory response are actively being pursued for MS therapy.^2,3,4,5,6 One growth factor associated with oligodendrocyte maturation, survival and dampening the immune response is growth-arrest specific protein 6 (Gas6). Gas6 is a secreted protein that is widely expressed in the central and peripheral nervous systems by endothelial cells and neurons, and is involved in numerous physiological and pathological functions including cell growth, survival and apoptosis.^{7,8,9,10,11,12} Gas6 binds and activates the TAM family of receptor tyrosine kinases consisting of Tyro3 (Rse/Dtk/Sky), Axl (Ufo), and Mer (Eyk).^{8,11,13,14,15} Many cell types express all three receptors and receptor activation can result from homophilic and heterophilic interactions.^16,17 Axl contains the major and minor Gas6 binding groove. Only the minor groove is conserved in Tyro3 and Mer and as a result, response to Gas6 is mediated in a concentration-dependent manner; Gas6 binding affinity is Axl>Tyro3>Mer.¹⁸We previously reported mRNA expression of Axl, Tyro3, and Mer receptors on human fetal oligodendrocytes and the ability of Gas6 to promote oligodendrocyte survival in vitro by activating Axl, resulting in Axl directly and indirectly recruiting phosphatidylinositol 3 kinase and activating the Akt pathway.^19,20 Moreover, we have shown that Gas6/Axl signaling through the Akt pathway can protect oligodendrocytes from tumor necrosis factor α (TNFα)-induced apoptosis.²¹ Down-regulation or deletion of Axl, even in the presence of Gas6, results in loss of protection against TNFα.²²During the relapse phase of relapsing-remitting MS, serum TNFα levels and TNFα mRNA are elevated.^23,24 TNFα is one of the major cytokines expressed in MS lesions.²⁵ TNFα is cleaved to its mature, soluble, secretable form by the matrix metalloproteinase (MMP) TNFα converting enzyme, also known as ADAM17.^26,27,28 MMPs, including ADAM17 and ADAM10 are involved in normal processes such as wound repair and tissue remodeling and are associated with disease states, including MS.^{29,30,31,32,33,34,35,36} Expression of ADAM17 is observed in acute and chronic active MS lesions, primarily in perivascular cuffs and cells morphologically resembling lymphocytes.³⁷ ADAM17 up-regulation in cerebrospinal fluid of MS patients is associated with inflammation and increased soluble TNFα.^37,38 ADAM10 is constitutively expressed on astrocytes in normal appearing white matter and on astrocytes and perivascular macrophages in MS lesions.^38,39,40,41 ADAM10 cleaves Axl, and ADAM17 cleaves Axl and Mer. Cleaved forms of receptors can result in internalization of the receptor and transport off the membrane for recycling. Cleavage can also result in shortened, soluble forms that act as a decoy to regulate the level of a growth factor at the membrane.^41,42Soluble forms of Axl and Mer can reduce the number of viable receptors for Gas6 binding and act as a decoy by sequestering Gas6 extracellularly; potentially a normal mechanism of receptor activation regulation.^40,41 During inflammation, soluble Mer has been reported to inhibit macrophage clearance of apoptotic cells.^41,43 Also, soluble Axl blocked the protective effect of membrane-bound Axl by inhibiting Gas6 induced tyrosine phosphorylation of Axl.⁴⁴ The binding of Gas6 to TAM receptors acts as an inhibitor of inflammation by inhibiting Toll-like receptor and cytokine receptor cascades.⁴⁴ Up-regulation of Axl and its subsequent interaction with interferon α and β receptors results in the expression of cytokine and Toll-like receptor inhibitors.^44,45 Thus, loss of Gas6 signaling, along with dysregulation of the balance between Gas6, Axl and Mer by increased extracellular levels of soluble Axl and Mer, might detrimentally impact the nervous system, especially in established (chronic active and chronic silent) lesions associated with MS. Chronic active MS lesions are characterized by ongoing demyelination, astrogliosis, macrophage and lymphocyte infiltration, astroglial hypertrophy, and oligodendrocyte hyperplasia.⁴⁶ Chronic silent MS lesions are characterized by the absence of actively infiltrating and inflammatory cells, oligodendrocyte loss and no evidence of ongoing demyelination.^46,47,48In this study, we investigated in chronic active and chronic silent MS lesions whether increased expression of soluble Axl and Mer was associated with increased expression of the MMPs ADAM17 and ADAM10, similar to previous studies that showed an association between increased ADAM17 and ADAM10 with TNFα in the CNS of MS patients.^37,38 We also investigated whether in lesions increased soluble Axl and Mer was associated with decreased Gas6, resulting in loss of the beneficial effects from activating membrane-bound Axl and Mer receptors.