Abstract
Changes in intracellular calcium concentration ([Ca2+]i) as well as in the phosphorylation state of proteins have been implicated in keratinocyte wound healing revealed in scratch assays. Scratching confluent HaCaT monolayers decreased the number of cells displaying repetitive Ca2+ oscillations as well as the frequency of their Ca2+-transients in cells close to the wounded area and initiated migration of the cells into the wound bed. In contrast, calyculin-A (CLA) and okadaic acid (OA), known cell permeable inhibitors of protein phosphatase-1 and 2A, increased the level of resting [Ca2+]i and suppressed cell migration and wound healing of HaCaT cells. Furthermore, neither CLA nor OA influenced how scratching affected Ca2+ oscillations. It is assumed that changes in and alterations of the phosphorylation level of Ca2+-transport and contractile proteins upon phosphatase inhibition mediates cell migration and wound healing.
Highlights
In mammalian cells changes in intracellular calcium concentration ([Ca2+]i) control a wide variety of functions, including proliferation, secretion, motility and contractility [1]
The serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) have been found to co-purify with protein kinase A (PKA) and IP3R, which is reminiscent of their interaction with RyR2 in heart muscle
Series of x-y images were recorded both on Fluo-4 loaded control and drug treated cultures, these cultures were scratched with a pipette tip and cells next to the wound were examined in the same way. 500 images with 500 ms intervals were taken from 3–6 different visual fields of each culture before and after scratching
Summary
In mammalian cells changes in intracellular calcium concentration ([Ca2+]i) control a wide variety of functions, including proliferation, secretion, motility and contractility [1]. Calcium ions underlying Ca2+ oscillations are released from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), and often spread through the cytoplasm as a regenerative Ca2+ wave [2]. This phenomenon is well-known in excitable cells, but some non-excitable cells, such as endothelial cells [3], osteoblasts [4], and chondrocytes [5] were shown to display calcium oscillations. The serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) have been found to co-purify with protein kinase A (PKA) and IP3R, which is reminiscent of their interaction with RyR2 in heart muscle. The ability of PP1 to dephosphorylate RyR was demonstrated in both skeletal and cardiac muscle [6], which could indicate that a similar complex exists in heart muscle, but in other cell types as well, with the involvement of RyR1 and/or IP3R
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