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First published online February 8, 2006
doi: 10.1242/10.1242/jcs.02781

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Activity of the hSPCA1 Golgi Ca²⁺ pump is essential for Ca²⁺-mediated Ca²⁺ response and cell viability in Darier disease

¹ INSERM U563, Purpan Hospital, Place du Dr Baylac, BP 2028, 31034 Toulouse CEDEX 3 and Université Paul Sabatier, 31062 Toulouse, France
² Department of Dermatology, University of California, 4150 Clement Street, San Francisco, CA 94131, USA
³ Department of Medical Genetics, Purpan Hospital, Place du Dr Baylac, 31059 Toulouse CEDEX 3, France
⁴ Dermatology Service, Department of Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94131, USA

View larger version (20K): [in a new window]   Fig. 1. SERCA2 protein is decreased in keratinocytes from all DD patients. Total protein extracts were analysed by immunoblotting using specific antibodies to SERCA2. Internal controls were GAPDH. The OD values represent densitometric analysis of the bands.

View larger version (24K): [in a new window]   Fig. 2. The Ca2+ response is preserved in DD keratinocytes. Normal and DD keratinocytes were cultured on glass coverslips in medium containing 0.06 mM Ca2+ until they were 50% confluent, then loaded with the Ca2+-sensitive dye Fura-2 (see Materials and Methods). The cells initially were superfused with control solution containing: 138 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 1.0 mM Na2HPO4, 0.06 mM CaCl2; 10 mM glucose, pH 7.4, 286 mOsm, then switched to this solution with 1.0 mM Ca2+ added (arrow). Although the baseline Ca2+ concentrations were much lower in DD keratinocytes, both normal and DD keratinocytes responded with a sustained increase in intracellular Ca2+ after exposure to raised extracellular Ca2+ levels. The absolute increase in intracellular Ca2+ was similar between DD and control keratinocytes, whereas the fold increase was significantly higher in DD keratinocytes (see Table 2).

View larger version (29K): [in a new window]   Fig. 3. Thapsigargin-releasable Ca2+ stores are decreased and capacitive Ca2+ influx is increased in DD keratinocytes. Normal and DD keratinocytes were cultured and loaded with Fura-2 as detailed in Fig. 1. Cells initially were superfused with solution containing: 138 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 1.0 mM Na2HPO4, 0.0 mM CaCl2, 0.05 mM EGTA, 10 mM glucose, pH 7.4, 286 mOsm. After equilibration, 500 nM thapsigargin was applied (black arrows). Although the resulting ER Ca2+ release substantially increased intracellular Ca2+ in normal keratinocytes, minimal Ca2+ release was noted in DD keratinocytes, consistent with depletion of the SERCA-dependent ER Ca2+ stores (Table 2). After recovery, the extracellular Ca2+ was increased to 0.06 mM (white arrows), initiating capacitive Ca2+ entry. Both normal and DD keratinocytes displayed robust capacitive Ca2+ entry; however, the capacitive Ca2+ entry was proportionately larger in DD keratinocytes (Table 2).

View larger version (54K): [in a new window]   Fig. 4. hSPCA1, but not PMCA, is increased in DD keratinocytes. Total protein extracts were analyzed by immunoblotting using specific antibodies to PMCA1 and PMCA4 and hSPCA1. Internal controls were GAPDH or ß-actin. The OD values result from the densitometric analysis of the bands. These data are representative of two separate experiments.

View larger version (32K): [in a new window]   Fig. 5. Inactivating ATP2C1 with siRNA blocks the Ca2+ response in DD keratinocytes. DD keratinocytes were cultured on glass coverslips until 40% confluent, then treated with ATP2C1 siRNA (see Materials and Methods). Forty-eight hours after treatment, at 50% confluence, cells were loaded with Fura-2 and intracellular Ca2+ was measured by the protocol detailed in Fig. 2. Ca2+ in the superfusing fluid was raised from 0.06 to 1.0 mM (black arrow). The Ca2+ response was blunted in DD keratinocytes treated with siRNA to ATP2C1 (top), whereas control DD keratinocytes responded normally to raised extracellular Ca2+ (bottom). The P value (Table 6) was calculated by a two-tailed t-test comparing vector control and siRNA. All values are expressed as mean ± s.d.

View larger version (25K): [in a new window]   Fig. 6. Pathways for Ca2+ signaling in normal and DD keratinocytes. Raised extracellular Ca2+ binds to a Ca2+ receptor (CaR), located in the plasma membrane, producing the second messenger inositol-1,4,5-trisphosphate (IP3). IP3 causes Ca2+ release from the endoplasmic reticulum (ER) and the Golgi by binding to IP3 receptors (IP3R). Emptying of intracellular Ca2+ stores activates Ca2+ influx through several pathways, including capacitive Ca2+ influx and influx through a Ca2+-permeable, Ca2+-activated non-selective cation channel (NSCC). In this drawing, calcium fluxes are denoted by solid arrows. Thick and thin arrows represent relative increases and decreases, respectively, in calcium flux between normal and DD keratinocytes: Ca2+ release from the ER decreases, whereas Ca2+ release from the Golgi and capacitive Ca2+ influx increase in DD cells relative to normal cells.

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