Increased sphingosine can enhance cell death/cell cycle arrest, previously reported in a canine model of DD (Muller, et al., 2006
), while increased levels of S1P promote cell growth and viability. The relative amounts of these lipids are determined by the activity of SPHK1, which metabolizes sphingosine to S1P; and SGPL1, which degrades S1P to phosphoethanolamine and hexadecanol (). Because abnormal sphingolipid signaling and DD share impaired cell differentiation, cell survival and calcium signaling, we first examined whether pharmacologically inactivating the ER Ca2+
ATPase SERCA2b could alter sphingolipid metabolism. We found that low concentrations of TG (100nM) increased sphingosine concentrations (). We were able to detect decreased sphingosine kinase mRNA expression after treatment with even lower TG concentrations (10-100nM) (). A similar decrease in SPHK1 expression was seen after inactivating SERCA2b with siRNA (). Both siRNA and TG were used to impair SERCA2 function in subsequent experiments. The dose of SERCA2b siRNA used for these experiments was chosen to reproduce the average 50% reduction in SERCA2b expression seen in DD patients (Foggia, et al., 2006
) (figure S1
). In the same paper, the authors report an average basal level of ER calcium for DD cells that is 60% lower than control as observed by transient cytosolic calcium increase after TG exposure. We observed that a similar decrease in ER calcium was obtained by treating cells with 10nM TG (Celli, et al., 2010
, and ) and we therefore used this concentration for the functional studies presented in this report.
Defects in ER Ca2+ Sequestration are Induced by ER SERCA2 Inhibition, and are Normalized by Inhibiting SGPL1
The ER Ca2+
store is important for normal keratinocyte signaling and differentiation (Callewaert, et al., 2003
; Foggia, et al., 2006
). Previous experiments demonstrated that early differentiation protein expression is inhibited by disabling the ER Ca2+
ATPase (Li, et al., 1995b
; Li, et al., 1995a
). Early differentiation proteins also are decreased in SERCA2 knockout mouse keratinocytes (Hong, et al., 2010
), and defects in E-cadherin localization have been reported in human DD (Tada and Hashimoto, 1998
). Decreased E-cadherin levels and/or trafficking may directly lead to decreased cell-to-cell adhesion via impaired formation of adherens junctions and may additionally impair IP3 mediated keratinocyte Ca2+
signaling because the E-cadherin complex formation in the membrane is essential for PLCγ1 activity (Xie and Bikle, 2007
; Xie, et al., 2009
). We therefore assessed whether modulating the sphingolipid pathway could reproduce or rescue defects in keratinocyte differentiation and E-cadherin expression or localization. We observed abnormal keratinocyte morphology and growth 24 hours (data not shown) and 48 hours after TG treatment (). Inhibition of SGPL1 with siRNA rescued these defects (). Western blotting showed that SERCA2 inhibition with TG also led to defects in involucrin protein synthesis and E-cadherin protein synthesis and processing. These defects could be normalized or ameliorated by inhibiting SGPL1 synthesis (). We found that SGPL1 inhibition was more effective in normalizing the defects in involucrin and E-cadherin expression caused by lower doses of TG (10 nM) than after higher (100nM) TG (data not shown), likely because the effects of SERCA2 inhibition became irreversible at higher doses. Treatment with exogenous S1P had no effect (Fig. S3
). Vitamin D and cannabinoid analogues also were ineffective (Fig. S3
Defects in Keratinocyte Morphology, Induced by SERCA2 Inhibition, are Normalized by Inhibiting SGPL1
Defects in Involucrin and E-cadherin synthesis and Processing Are Normalized by Inhibiting SGPL1
The additional, higher molecular weight band seen on immunoblots after TG treatment suggested that E-cadherin intracellular processing and localization might also be disrupted when ER Ca2+
stores were depleted, similar to what has previously been reported for DD (Hakuno, et al., 2000
). To test this, we localized E-cadherin first using an antibody that selectively binds to the extracellular epitope (). E-cadherin normally responds to raised extracellular Ca2+
by forming continuous, fine intercellular strands (). TG treatment lead to a dose-dependent disruption in these cell-to-cell contacts, with E-cadherin localization becoming coarse and irregular at very low (10nM) TG concentrations (), and discontinuous at 100 nM TG concentrations (data not shown). SGPL1 inhibition normalized E-cadherin localization at 10 nM TG (), but only partially rescued TG-induced E-cadherin disruption at the 100 nM dose (not shown). Treatment with scrambled siRNA had no significant effect (). These data suggest that E-cadherin is not properly localized to the plasma membrane after SERCA2 inhibition.
Defects in E-cadherin localization Are Normalized by Inhibiting SGPL1
To test whether SERCA2 inhibition lead to defective E-cadherin, we next localized E-cadherin using an antibody that binds to an intracellular E-cadherin epitope. After raising extracellular Ca2+, the vast majority of E-cadherin was found at cell-to-cell junctions in normal keratinocytes () with only punctuate foci of intracellular E-cadherin seen. After treatment with 10 nM TG, extracellular E-cadherin again appeared irregular and beaded (). In addition, perinuclear aggregates of E-cadherin also were seen (), suggesting that ER Ca2+ depletion leads to intracellular E-cadherin accumulation. This process also was dose-dependent, and the vast majority of E-cadherin was localized intracellularly after treatment with 100 nM TG (not shown). As above, inhibition of SGPL1 was able to normalize E-cadherin localization in keratinocytes treated with 10 nM TG (), but only partially rescued keratinocytes treated with the higher 100 nM TG (not shown). Treatment with scrambled siRNA did not rescue the defects caused by TG treatment ().
Downregulation of SERCA2b expression by siRNA resulted in a similar pattern of intracellular and extracellular E-cadherin staining ( respectively) that could be normalized by SGPL1 siRNA treatment (). Treatment with a scrambled siRNA sequence had no effect ().
Defects in E-cadherin localization following downregulation of SERCA2b expression with siRNA Are Normalized by Inhibiting SGPL1
These studies demonstrate that E-cadherin requires normal ER Ca2+ stores to properly localize to the plasma membrane, and that the defects seen in cell-to-cell adhesion in DD may be due to defective E-cadherin localization.
To further investigate the effects of SERCA2 inactivation on cell to cell junctions, we assessed the effect of 10 nM TG on desmoplakin (DP) localization, which also is disrupted following SERCA2 inactivation (Hobbs, et al., 2011
). In agreement with this previous report, we also observed DP localization at the cell to cell borders within 3 hours after raising extracellular Ca2+
in control cells (). 48 hours after raising extracellular Ca2+
, DP was almost entirely localized to cell-to-cell borders (). Like E-cadherin, TG disrupted DP localization in a dose-dependent manner. Cells treated with 10 nM TG are shown in , and cells treated with 100 nM TG are shown for comparison in Fig. S5
. DP staining was mostly intracellular in TG-treated cells. Defects in DP staining and gaps between cells were noted at both the 3 and 48 hours time points ( respectively). Treatment with SGPL1 siRNA completely rescued DP localization 48 hours after calcium switch, but only partially at the earlier 3 hour time point ( respectively). Treatment with scrambled siRNA did not rescue the abnormal DP localization (). SGPL1 siRNA also partially rescued DP localization in cells treated with 100nM TG (Fig. S5
Defects in Desmoplakin (DP) localization, Caused by SERCA2 inactivation with TG, Are Normalized by Inhibiting SGPL1
We used the ER-targeted Ca2+
sensor D1ER to monitor ER Ca2+
depletion after SERCA2 inhibition, and to assess whether SGPL1 inhibition could ameliorate this loss. and present the data as average FRET ratios and average calcium concentrations respectively. A decrease in FRET ratio indicates lower Ca2+
concentrations. FRET ratios were converted into calcium concentration following the protocol in (Rudolf, et al., 2006
). siRNA transfection alone, with either a scrambled sequence or SGPL1 siRNA, leads to a small decrease in ER calcium levels. In agreement with a previous report (Celli, et al., 2010
), treatment with 10nM thapsigargin caused a dramatic drop in ER calcium concentration in both untreated and scrambled siRNA transfected samples. However, treatment with 10nM TG had no significant effect on ER Ca2+
in cells pretreated with SGPL1 siRNA (). TG treatment produced more than a 50% drop in ER Ca2+
concentration in both untreated keratinocytes and scrambled siRNA-transfected keratinocytes (). In contrast, SGPL1 inhibition prevented this TG- induced Ca2+
loss (). These data suggest that modulating sphingosine metabolism may normalize TG-induced abnormalities in keratinocyte differentiation and cell-to-cell adhesion by mitigating ER Ca2+
loss after SERCA2 inactivation. 100 nM TG almost completely emptied ER Ca2+
stores and could not be rescued by SGPL1 inhibition (data not shown). SGPL1 inhibition did not change SERCA2 protein levels or SPHK1 mRNA expression, suggesting that Ca2+
was normalized by a mechanism independent of SERCA2 or SPHK1 synthesis (Fig. S1 and S2
This report demonstrates that inhibiting SGPL1 rescues ER Ca2+
depletion-induced defects in keratinocyte differentiation and adhesion, and restores ER Ca2+
stores depleted by SERCA2 inhibition. How sphingosine metabolism and SERCA2 Ca2+
signaling interact in directing keratinocyte adhesion and differentiation is not yet well defined. Certainly, the actions of exogenous S1P in controlling intracellular Ca2+
have been studied extensively. However, we found that exogenous S1P was ineffective in rescuing adhesion and differentiation defects caused by SERCA2b inactivation (Fig. S3
). These data suggest that generation of intracellular S1P is essential for rescuing the DD phenotype. Alternatively, extracellular S1P-stimulated Ca2+
release from the ER may be blunted when these stores already are depleted due to SERCA2 dysfunction. Exposure to increased intracellular S1P levels, on the other hand, has been shown to increase calcium levels in TG sensitive stores and to mobilize calcium from these stores into the cytosol, independently of the plasma membrane G-protein coupled S1P receptor (Claas, et al., 2010
; Meyer zu Heringdorf, et al., 2003
In a recent paper, Calloway et al (Calloway, et al., 2009
) found that treatment of RBL mast cells with sphingosine and its positively charged analogues inhibited the Ca2+
influx via SOCE usually seen after TG treatment, by flipping to the inner leaflet of the plasma membrane and preventing Ora1/CRACM1 from interacting with STIM1. Thus, SERCA2b inactivation might cause not only ER Ca2+
depletion, but also lead to increased intracellular sphingosine, via SPHK1 inhibition. The increased sphingosine then might interfere with Ora/CRACM1/STIM interaction, exacerbating the low ER Ca2+
caused by SERCA2b loss.
We chose to study the effects of ER calcium depletion and SGPL1 down-regulation on E-cadherin because this protein has both a structural function and a role in calcium signaling (Xie and Bikle, 2007
; Xie, et al., 2009
). Moreover, calcium dependent E-cadherin aggregation at cell-to-cell adhesion points is known to be perturbed in DD (Tada and Hashimoto, 1998
). In contrast to a previous study (Hobbs, et al., 2011
) our experiments demonstrated that E-cadherin localization was disrupted after SERCA2 inhibition. This discrepancy may be explained by the longer timecourse (24–48 hours vs. 3 hours) used in our studies, based on previous findings that E-cadherin continues to aggregate and complex at cell-to-cell junctions for at least 7–20 hours (Vasioukhin, et al., 2000
). Immunofluorescence micrographs of SERCA2 siRNA treated cells harvested 3 hours after calcium switch do not show significant defects in E-cadherin localization (Fig. S4
) as compared to the longer time points, thus lending support to this hypothesis.
SGPL1 inhibition also normalized desmoplakin (DP) trafficking, which has been implicated in DD (Dhitavat, et al., 2003
; Hobbs, et al., 2011
). These studies suggest that modulating the sphingolipid pathway may ameliorate the defective cell-to-cell adhesion that is a hallmark of DD.
We studied cells treated with thapsigargin or siRNA against SERCA2 in lieu of DD keratinocytes, as previous studies conducted on DD keratinocytes revealed a wide heterogeneity in calcium mobilization in these cells (Foggia, et al., 2006
). While further investigation on the effects of SGPL1 inhibition in DD cells is needed, we show here that reversing SERCA2 inhibition caused by thapsigargin or SERCA2 siRNA ameliorates the junctional and differentiation defects that are known to underlie DD pathology.