Pemphigus IgG, but Not Bullous Pemphigoid IgG, Causes a Transient Increase in Intracellular Calcium and Inositol 1,4,5-Triphosphate in DJM-1 Cells, a Squamous Cell Carcinoma Line

Abstract

It is still unclear what kinds of mechanisms are involved in blister formation after antibodies bind to the antigens in pemphigus and bullous pemphigoid. The effects of IgGs from pemphigus vulgaris, pemphigus foliaceus, and bullous pemphigoid sera on intracellular calcium concentration ([Ca++]i) and inositol 1,4,5-trisphosphate were examined in a human squamous cell carcinoma cell line (DJM-1 cells) and in cultured human keratinocytes to clarify whether signal transduction via calcium is involved. IgGs were purified with protein A affinity column from the sera of five pemphigus vulgaris patients, three pemphigus foliaceus patients, eight bullous pemphigoid patients, and 14 normal volunteers. Keratinocytes were cultured in Eagle's minimum essential medium containing 1.8 mM Ca++ and loaded with fura-2/AM, followed by addition of the IgGs. Subsequently, [Ca++]i was determined by measuring the fluorescence ratio (F340/F360) with videomicroscopy. Pemphigus IgGs (seven of eight cases) induced a rapid and transient increase in [Ca++]i in both the cells, whereas a [Ca++]i increase was caused by very few IgGs from bullous pemphigoid (one of eight cases) and normal sera (two of 14 cases). The pemphigus IgG-induced transient [Ca++]i increase was not affected by chelating extracellular Ca++ with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetracetic acid. In addition, monoclonal antibodies acid. In addition, monoclonal antibodies against 180-kD and 230-kD antigens did not exert this change. Pemphigus IgGs that caused a [Ca++]i increase induced rapid and transient production of inositol 1,4,5-trisphosphate, peaking at 20 seconds. These findings suggest that IgG from pemphigus induces Ca++ mobilization by inositol 1,4,5-trisphosphate from internal stores, and that mechanisms of antibody-transmitted signaling in pemphigus may differ from those in bullous pemphigoid.

Novel Mechanisms of Target Cell Death and Survival and of Therapeutic Action of IVIg in Pemphigus

“…64 An increase of calpain activity in keratinocytes treated with PVIgGs observed in this study is in keeping with observations that binding of PVIgG to keratinocytes causes a transient increase in intracellular free Ca 2ϩ , activates phospholipase C, stimulates production of inositol 1,4,5-trisphosphate, induces activation and translocation of protein kinase C from the cytosol to the particulate/cytoskeleton fractions, and increases the phosphorylation status of cellular proteins. 4,30,[65][66][67][68][69][70] Furthermore, it has been recently reported that inhibitors of tyrosine kinases, phospholipase C, calmodulin, and the serine/threonine kinase protein kinase C prevented PVIgG-induced acantholysis in vivo. 71 TNF-␣ has been shown to activate calpains because of a rise in the concentration of intracellular free Ca 2ϩ .…”

Desmosomal Cadherins and Signaling: Lessons from Autoimmune Disease

“…Pemphigus antibodies were initially thought to disrupt Dsg trans-interaction but later have been shown to also exert their pathological effects by triggering intracellular signaling pathways Seishima et al, 1995). Direct steric inhibition apparently mimics the so-called tryptophan-swap between interacting Dsg3 molecules which hampers cis-and trans-interaction (Di Zenzo et al, 2012;Spindler et al, 2013;Yamagami et al, 2010).…”

“…Many signaling molecules have been associated with pemphigus pathogenesis and shown to contribute to loss of cell adhesion in a variety of model systems (Getsios et al, 2010;Waschke, 2008). These include phospholipase C, Ca 2 ϩ , and protein kinase C (PKC) Osada et al, 1997;Seishima et al, 1995) as well as plakoglobin regulating the expression of Myc (Caldelari et al, 2001;Williamson et al, 2006). Rho GTPases act as molecular switches in actin dynamics (Gliem et al, 2010;Waschke et al, 2006).…”

Desmosome Signaling

“…Proposed mechanisms for PV IgG-induced keratinocyte detachment include (a) proteinase activation, (b) steric hindrance, and (c) activation of transmembrane signaling that down-regulates cell-cell adhesion (7)(8)(9)(10)(11). Previous work has suggested that PV IgG may activate intracellular signaling events; however, the precise nature and biological consequences of these events and their relationship to the mechanism of PV IgG-induced acantholysis remain unknown (7,8).…”

“…Previous work has suggested that PV IgG may activate intracellular signaling events; however, the precise nature and biological consequences of these events and their relationship to the mechanism of PV IgG-induced acantholysis remain unknown (7,8). The specificity of anti-dsg3 antibodies in PV patient sera enabled us to utilize this reagent to initiate changes in desmosome structure and look for activation of signaling and the relationship of these signaling events to the mechanism of acantholysis.…”