Upregulation of the calcium-dependent protease, calpain, during keratinocyte differentiation

Abstract

Calpain is a ubiquitous neutral calcium-activated thiol protease that is implicated in various cellular functions including exocytosis, cell fusion, apoptosis and proliferation. The calpain system is composed of the enzymes mu-calpain and m-calpain and their endogenous inhibitor, calpastatin. We employed the spontaneously immortalized human HaCaT keratinocytes, which retain their ability to differentiate in vitro and in vivo, to study the modulation of the calpain system during keratinocyte differentiation. The cellular levels of keratinocyte differentiation markers and of the components of the calpain system were monitored by immunoblotting. Three established differentiation stimuli: increase in cell density as a function of time in culture, elevation of extracellular calcium concentration and exposure to 1,25-dihydroxyvitamin D3 enhanced the expression of the three keratinocyte differentiation markers keratin 10, involucrin and transglutaminase. The differentiation of HaCaT cells was accompanied by elevation of the components of the calpain system, although the pattern of increase varied according to the specific differentiation stimulus. A higher increase in calpains as compared with the increase in calpastatin suggests an increase in net calpain activity during differentiation. Such an increase may play a part in the differentiation process itself and/or in the regulation of key events in differentiating keratinocyte metabolism.

Sedimentation field-flow fractionation separation of proliferative and differentiated subpopulations during Ca2+-induced differentiation in HaCaT cells

“…5). Results obtained for LC-TP and HC-TP were similar to those observed previously: (1) LC cells were mainly in G0/G1, due in part to confluence, which induced differentiation [12,13,15]; (2) whereas HC cells accumulated in S/G2-M [14]. The cell cycle profile of HC-F2 (Fig.…”

“…Like NHKs, HaCaT differentiation is induced either with confluence, when cells are cultured in a calcium free medium [12][13][14][15] or with Ca 2+ concentrations superior to 1 mM [62,63]. In both cell types, Ca 2+ -induced differentiation is observed by an increased expression of differentiation markers, such as K1, K10 and involucrin [5][6][7][8][9][10].…”

Deimination of Human Filaggrin-2 Promotes Its Proteolysis by Calpain 1

“…The protease has been detected in the upper part of the epidermis (43), and its expression has been shown to be up-regulated during keratinocyte differentiation in vitro (44). In our studies, in vitro cleavage assays revealed that calpain 1 was able to directly process a fragment of FLG2 consisting of B-type repeats B7-B10 into multiple peptides detected by both immunoblotting and mass spectrometry.…”

Post-translational control of Myc function during differentiation

“…59 In these tissues, Myc-nick may be involved in mediating at least some of the functions attributed to the myc gene in terminal differentiation and tissue maintenance. Furthermore, during keratinocyte differentiation calpains are activated 28 and there is an increase in tubulin acetylation, 62 suggesting a correlation with Myc-nick production and function. In a keratinocyte model for the autoimmune disease Pemphigus vulgaris, a smaller form of Myc, with a size corresponding to that of Myc-nick, was detected predominantly in the cytoplasm.…”

“…While hyperactivation of calpains is associated with increased tumorigenesis, 27 regulated increases in calpain activity are essential for terminal differentiation of multiple cell types. Calpains are upregulated and activated during differentiation of keratinocytes, 28 hematopoietic 29 and muscle cells, 30,31 among others. Importantly, mutations in the muscle-specific calpain 3, which impair its proteolytic activity, are the sole cause of limb girdle muscular dystrophy 2A (LGMD2A).…”