|Home | About | Journals | Submit | Contact Us | Français|
Endoplasmic reticulum (ER) Ca2+ depletion, previously shown to signal pathologic stress responses, has more recently been found also to trigger homeostatic physiologic processes such as differentiation. In keratinocytes and epidermis, terminal differentiation and barrier repair require physiologic apoptosis and differentiation, as evidenced by protein synthesis, caspase 14 expression, lipid secretion, and stratum corneum (SC) formation.
To investigate the role of Ca2+ depletion induced ER stress in Keratinocytes differentiation and barrier repair in vivo and in cell culture.
The SERCA2 Ca2+ pump inhibitor Thapsigargin (TG) is used to deplete ER Calcium both in cultured Keratinocytes and in mice. Levels of the ER stress factor XBP1, loricrin, Caspase 14, lipid synthesis and intracellular Ca2+ are compared after both TG treatment and barrier abrogation.
We show here that these components of terminal differentiation and barrier repair are signaled by physiologic ER stress, via release of Stratum Granulosum (SG) ER Ca2+ stores. We first find that keratinocyte and epidermal ER Ca2+ depletion activate the ER-stress-induced transcription factor XBP1. Next, we demonstrate that external barrier perturbation results in both intracellular Ca2+ emptying and XBP1 activation. Finally, we show that TG treatment of intact skin does not perturb the permeability barrier, yet stimulates and mimics the physiologic processes of barrier recovery. This report is the first to quantify and localize ER Ca2+ loss after barrier perturbation and show that homeostatic processes that restore barrier function in vivo can be reproduced solely by releasing ER Ca2+, via induction of physiologic ER stress.
The Endoplasmic Reticulum (ER) responds to perturbations in its luminal environment by initiating ER stress responses. Although the ER stress response was originally described as a compensatory response to pathological perturbations, more recently it has become clear that this response also participates in normal cellular differentiation. The unfolded protein response (UPR), one component of the ER stress response, has been shown to mediate differentiation in pancreas 1 and plasma cells 2(and reviewed in 3) via activation of the inositol-requiring 1 (IRE-1) sensor, which in turn generates the active (spliced) form of the transcription factor XBP1.
Keratinocytes and epidermis form a unique model to study ER stress, particularly ER stress due to ER Ca2+ depletion. Profound ER Ca2+ depletion, caused by a variety of mutations in the ER Ca2+ ATPase ATP2A2 (protein SERCA2), leads to Darier’s disease, a skin condition characterized by defective keratinocyte differentiation, abnormal keratinocyte apoptosis, and impaired cell-to-cell adhesion known as acantholysis (4–6. When SERCA2 is inactivated in mice, altered differentiation leads to development of squamous cell carcinomas in these animals’ skin 7. Conversely, mild or physiologic ER stress seems to enhance differentiation, particularly differentiation characteristic of later, terminal differentiation 8,9. Proteins associated with the ER XBP1-mediated stress pathway also are seen in the differentiated upper layers of normal epidermis, but not in hyperplastic benign (psoriasis) or malignant (squamous cell carcinoma) epidermis 9, and activated XBP1 is upregulated after UVB treatment of keratinocyte HaCaT cells 10.
We therefore hypothesized that mild, physiologic ER stress responses might drive normal keratinocyte differentiation, especially the terminal differentiation seen in the upper epidermis in response to barrier perturbation. Specifically, we examined whether decreases in ER Ca2+ concentrations, known to induce ER stress in keratinocytes 9 might mediate well-defined aspects of terminal differentiation seen after barrier perturbation in the uppermost viable layer of the epidermis, the Stratum Granulosum (SG).
Ca2+ is known to modulate both keratinocyte and epidermal differentiation. Experimental manipulation of Ca2+ in the upper epidermis regulates both the expression of specific epidermal differentiation proteins 11,12, and lipid secretion from cells of the outer SG 13–15, leading to epidermal barrier recovery. Acute barrier perturbation, either by physical removal of the SC or by solvent extraction of lipids, sets in motion a rapid sequence of events which together lead to epidermal barrier restoration and repair (reviewed in 16 and 17. Within 15–30 minutes, most preformed lamellar body contents are secreted from the SG into the spaces between the outer SG and SC. Concurrently, caspase 14, which directs terminal differentiation (physiological apoptosis), is expressed and activated in the outermost SG 18. These SG cells then lose their organelles and plasma membranes (transitional cells) and form anucleate cells bounded by cornified envelopes (SC). These processes, along with permeability barrier homeostasis, are modulated by experimental interventions that change epidermal Ca2+ 13–15,19.
Although initial experiments implicated extracellular Ca2+ fluxes in responses to barrier perturbation 11,12, more recent experimental evidence suggests that intracellular Ca2+ stores also play a pivotal role in terminal differentiation. Genetically modified mice in which capacitive Ca2+ entry is impaired by deleting the store-operated Ca2+ permeable channel Trpv6 display abnormal epidermal Ca2+ gradients 20. Moreover, in a recent study we found that the vast majority of the Ca2+ in the SG is found in intracellular stores rather than in extracellular spaces 21. In the current study, we investigated whether ER stress mechanisms, activated by ER Ca2+ loss, underlie epidermal permeability barrier homeostasis. This report is the first to show that barrier repair processes in vivo can be reproduced simply by depleting ER Ca2+. While profound loss of ER Ca2+ results in a pathologic skin condition known as Darier’s disease, physiologic ER Ca2+ release, resulting in ER stress, seems to underlie barrier homeostasis.
While TG is a specific inhibitor of the ER Ca2+ ATPase, high concentrations of this agent can have non-specific or toxic effects (Sorin et al 1997). In order to assess the effects of specifically depleting the ER Ca2+ stores, we first determined the concentrations of TG needed to deplete the keratinocyte ER Ca2+ pool. We found a long-lasting, concentration dependent ER Ca2+ depletion after TG treatment, at low concentrations of 10–100 nM (Figure 1A and B). We next assessed whether keratinocyte ER Ca2+ depletion using these TG concentrations activates the ER protein XBP1. ER stress results in elevated levels of XBP1 and XBP1 activation by alternative splicing (Yoshida et al, 2001; Hirota et al, 2006). Active XBP1 was upregulated, in a dose-dependent manner, when cultured human keratinocytes were exposed to 10 and 50 nM TG (Figure 2).
Since XBP1 was activated when ER Ca2+ stores were depleted in cultured human keratinocytes, we next tested whether XPB1 also is upregulated by ER Ca2+ depletion in epidermis. We found a dose dependent upregulation of the active form of XBP1 following topical TG applications to mouse flank skin (Figure 3). Double tailed t-test demonstrated statistically significant differences between the TG treated samples vs. control and untreated samples (p<0.003 for 50 and 100 nM TG). To confirm that upregulation of XBP1 after TG application was due specifically to ER Ca2+ release, as opposed to a non-specific injury response to barrier perturbation, we monitored barrier homeostasis using transepidermal water loss, a sensitive indicator of barrier injury. Epidermal permeability barrier homeostasis, as measured with TEWL, did not differ in untreated animals vs. animals treated with vehicle or TG (data not shown).
We next used a minimally invasive imaging approach, 21, and also see Materials and Methods), to quantify Ca2+ depletion from intracellular stores following barrier abrogation. We measured the fluorescence lifetime changes of the calcium sensitive dye Calcium Green 5N 21 to quantify SG intracellular Ca2+ in ex vivo skin before and after barrier perturbation. A clear difference in calcium levels is observed between the intra and extracellular calcium levels in the SC (Figure 4A), while the extracellular space falls below the microscope resolution in the granular layer (Figure 4B). Using this approach we previously showed that most Ca2+ in the epidermis, especially in the SS and SG, is localized intracellularly 21. Consistent with this previous report, prior to acute barrier disruption, we found long lifetime decays, indicative of high intracellular Ca2+ concentrations, within SG keratinocytes (Figure 4 B). As reported previously in 21, the Ca2+ concentrations measured, >20 µM, suggest that this signal derives primarily from intracellular stores such as the ER.
Next, we quantified changes in intracellular Ca2+ concentrations after epidermal permeability barrier abrogation (Figure 4C) by tape stripping (note that most of the SC is removed after tape stripping). The lifetime measured in the SG of tape stripped epidermis (Figure 4 C) became dramatically shorter than in unperturbed skin (Figure 4B), indicating a precipitous drop in Ca2+ concentration after acute barrier abrogation, from >20 µM to <1.5 µM. We previously showed that the intracellular space occupies 98% of the volume in normal SS and SG epidermis 21. Consistent with this finding, most of the Ca2+ lost from the SG was derived from the intracellular stores. These results quantify the dramatic loss of SG Ca2+ seen after barrier perturbation, and further localize this loss to intracellular Ca2+ stores such as the ER.
The distributions of calcium concentrations at different epidermal strata before and after tape stripping are shown in Figure 4 D and E respectively.
We next assessed whether epidermal permeability barrier abrogation leads to ER stress, by measuring activated XBP1 after barrier abrogation. Consistent with our hypothesis, active XBP1 mRNA was upregulated in mouse epidermis by both chemical (acetone lipid extraction) and mechanical (tape stripping) barrier abrogation (Figure 5). Statistical analysis with a double tailed Students t-test showed significant differences between XBP1 levels from unperturbed vs. perturbed samples (p<0.0001). Taken together, the experiments detailed above suggest that epidermal permeability barrier homeostasis is mediated by ER stress, induced by changes in SG ER Ca2+.
To test whether the epidermis senses barrier perturbation and initiates barrier homeostatic mechanisms via ER Ca2+ depletion and stress, we tested whether depleting ER Ca2+ stores, without perturbing the epidermal barrier, would nevertheless stimulate and mimic the physiologic processes of barrier recovery and terminal differentiation.
Barrier recovery and terminal differentiation consist of simultaneous, parallel processes. Epidermal Ca2+ has been shown to coordinately regulate both lamellar body secretion and terminal differentiation (i.e. cornified envelope formation, loss of cellular organelles, and synthesis of differentiation-specific proteins) 22–24. In addition, epidermal terminal differentiation also is directed by activation of caspase 14, a cysteinyl aspartate-specific proteinase (reviewed in 25). To determine which processes are controlled specifically by ER Ca2+ emptying, we treated normal hairless mice with a single topical application of a low concentration (100 nM) of TG, applied to unperturbed flank skin. By two hours after application, we found that TG treatment increased both epidermal caspase 14 expression and activation (Figure 6A and 6B) and loricrin expression (Figure 7), consistent with enhanced differentiation. As noted above, epidermal permeability barrier homeostasis, as measured by the kinetics of transepidermal water loss (TEWL), did not differ in untreated animals vs. those treated with vehicle vs. those treated with TG.
EM images showed that release of ER Ca2+ stimulated a burst of lamellar body secretion around all surfaces of the outermost granular cells (Figure. 8A vs. 8B), which was paralleled by a wave of transitional cell formation, indicated by loss of intracellular organelles (Fig. 8B-TC). However, none of these newly-generated transitional cells display nascent cornified envelopes, which instead appeared only 2–3 cell layers higher, where these transitional cells transformed further into corneocytes (Figs. 8B, broken arrow). All preformed lamellar bodies were secreted after TG treatment, as evidenced by the lack of entombed organelle contents within corneocytes (see also below).
To further characterize the relationship between lamellar body secretion and transitional cell formation, we next examined this skin with ultrastructural cytochemistry, using a lamellar body content marker, acidic lipase 26, to detect the earliest stages of secretion. As seen in Figure 8C, TG treatment stimulated release of lamellar body contents beneath the outermost granular cell layer, immediately prior to TC formation, evident in images from adjacent granular cells that display features of TC (Fig. 8B and 8F). Vehicle-treated control images for comparable levels of SG are seen for comparison in Figs. 8A and 8D. Together, these results suggest that Ca2+ release from ER stimulates lamellar body secretion, which in turn induces formation of transitional cells, prior to the appearance of cornified envelopes. Yet, cornified envelopes eventually appeared, and cornified envelope thickness increased in SC keratinocytes, indicative of enhanced terminal differentiation and SC formation (table 1). This sequence of events occurs too rapidly to be delineated after barrier perturbation, but is clearly seen when these processes are separated using an agent that stimulates ER Ca2+ depletion alone.
These studies demonstrate that ER Ca2+ loss, induced both by external barrier perturbation and by pharmacologic inactivation of the ER Ca2+ ATPase, lead to ER stress and a cascade of physiologic processes that maintain epidermal barrier homeostasis and lead to terminal differentiation. TG usually is used at concentrations up to 1 µM to empty ER Ca2+ stores 9. The concentrations used in this study were considerably lower, in the 10 –100 nM range. As shown in Figures 1 and and2,2, these concentrations were effective in lowering ER Ca2+ and activating XBP1, consistent with previously published reports that showed TG concentrations in this range altered keratinocyte morphology or differentiation (Li et al 1995; Jones and Sharp 1994).
Since very low (10 nM) concentrations of TG induce both ER Ca2+ loss and XBP1 activation, it seems likely that any significant loss of ER Ca2+ will result in ER stress. It previously has been shown that ER stress induced by agents, such as tunicamycin, that do not directly act on the keratinocyte ER Ca2+ ATPase also enhance terminal differentiation 9. Whether these agents also stimulate processes that mimic barrier repair, and whether it is possible to induce ER stress without changing the ER Ca2+ concentration, will be the subject of future studies.
In these studies, we showed that ER stress induced by ER Ca2+ release results in lamellar body secretion, and that increased secretion is paralleled by the immediate formation of transitional cells. Since these transitional cells still lack cornified envelopes, these results strongly suggest that lamellar body secretion is the initial trigger for terminal differentiation, independent of cornified envelope formation, which occurs subsequent to transitional cell formation. ER Ca2+ release could thus ensure that lamellar body contents are secreted prior to cornification. In turn, secreted lamellar body contents could effectively seal off SG cells, triggering terminal differentiation.
Mutations in the ER Ca2+ ATPase, encoded by the gene ATP2A2, cause Darier’s disease (DD) 4. The ER Ca2+ store is important for normal keratinocyte signaling and differentiation 27–29 6. Previous experiments testing the effects of TG in cultured mouse keratinocytes demonstrated that early differentiation marker expression were inhibited, while expression of later markers, such as loricrin, were enhanced 8,30. Expression of early differentiation markers is premature in DD 5, but decreased in SERCA2 knockout mouse keratinocytes (Hong et al 2009). Changes in later differentiation markers have not been described in DD. These results elucidate a novel Ca2+ signaling mechanism in skin. Modulating SG ER Ca2+ might be useful to treat diseases in which late differentiation or barrier function is impaired.
Primary normal human keratinocytes were isolated from newborn foreskins, and a mixture of first passage cells in 0.03 mM Ca2+ EpiLife (Cascade Biologics) media plated in 6-well plates to 80% confluence and incubated at 37°C with 5% CO2 were used for all cell culture studies. Keratinocytes were treated with 10 nM and 50 nM thapsigargin (Sigma-Aldrich) for 2 hours and washed with fresh media. RNA was isolated from cells using Qiagen’s RNeasy kit.
Truncal skin samples were obtained from the Dermatology surgical units under protocols approved by the University of California, San Francisco and San Francisco Veterans Affairs Medical Center and in accordance with the principles expressed in the Declaration of Helsinki. A total of 4 skin samples from three different patients where used. Two of the samples from two different individuals were tape stripped 40 times immediately following excision and two were used as unperturbed controls. Each sample was submerged in a 50 µM solution of CG5N in 0.06 mM [Ca+2] Epilife (Cascade, Oregon) so that both the Stratum Corneum and the dermal side were in contact with the dye solution, and incubated overnight at 4 °C to insure dye penetration in the deeper layers of the tissue. About 2 hours prior to imaging, the samples were rinsed in dye free Epilife in order to remove the excess dye and placed with the dermal side in contact with media. The specimens were mounted with the Stratum Corneum in contact with a glass coverslip and secured on the stage of an inverted Zeiss Axiovert 200 microscope (Zeiss, Germany).
Normal primary human keratinocytes were plated in glass bottom chambers in Keratinocyte Growth Media (Clonetics) with 0.03 mM Ca2+ , to allow for efficient transfection of the D1ER construct 31. Cells were transfected with D1ER (gift of Prof. Tsien’s laboratory) using Mirrus Keratinocyte Transit lipid reagent, according to the manufacturer’s protocol. 24 hours after transfection, cells were treated with TG, in a final concentration of 10 nM or 100 nM. After one hour, the cells were washed and transferred to Keratinocyte Growth Media containing 1.2 mM Ca+2 , to allow for efficient cell attachment and growth. Dual channel fluorescence measurements were performed 24 hours later using a Zeiss LSM Meta confocal system. Calibration of the Calcium sensitivity range of the probe and conversion of the FRET ratio into Calcium concentrations was performed following the protocol in 32.
Mice were treated under protocols approved by the University of California, San Francisco and San Francisco Veterans Affairs Medical Center. Normal hairless mice were anesthetized with 40mg/mL chloral hydrate and received dorsal flank treatments with a vehicle of 70% propylene glycol and 30% ethanol mixture or 100 nM thapsigargin in the vehicle. 2 hours later mice were euthanized and skin biopsies were harvested. Samples for electron microscopy were placed immediately in modified Karnovsky’s fixative according to previous protocol 11,12. Samples for immunohistochemistry were placed immediately in formalin (Fisher Scientific). Samples for RNA isolation were placed in Qiagen’s RNA Later solution and frozen at −80°C. Skin samples for protein and RNA isolation were placed in PBS supplemented with protease inhibitors and underwent heat separation at 56°C for 2 minutes 33. Epidermis was removed from dermis, washed twice with cold PBS/inhibitors, homogenized, and placed in RIPA Buffer supplemented with protease inhibitors. Protein lysates were harvested. Epidermis for RNA isolation was prepared as described above, homogenized, and RNA was isolated according to SV Total RNA Isolation kit (Promega) protocol. Electron microscopy and immunohistochemistry were performed using previously published protocol 11,12.
All cDNA was generated using Roche Applied Science’s Transcriptor first strand cDNA synthesis kit. Quantitative PCR (QPCR) was performed using TaqMan fast universal PCR master mix and the ABI 7900HT real-time PCR machine (Applied Biosystems) and target genes were normalized to GAPDH (primers and dual-labeled probes from Applied Biosystems). Human XBP1 primers and probes used were: forward 5’-aagccaaggggaatgaagt-3’, reverse 5’-ccagaatgcccaacaggata-3’, active form probe 5’-gctgagtccgcagcaggtgcag-3’, inactive form probe 5’-agcactcagactacgtgcacct-3’. Mouse Xbp1 primers and probes used were: forward 5’-gatcctgacgaggttccaga-3’, reverse 5’-atgttctggggaggtgacaa-3’, active form probe 5’-agtccgcagcaggtgcaggc-3’, inactive form probe 5’-cagactatgtgcacctctgcagca-3’. Data were analyzed using the relative standard curve method. ANOVA test was used to assess statistically significant differences in XBP1 activation between perturbed and unperturbed samples.
Cell culture and heat separated epidermis samples were homogenized, and proteins were isolated using RIPA buffer (Sigma-Aldrich) containing protease inhibitors (Roche Applied Science). Protein quantification for equal loading was made with Thermo Scientific Pierce’s BCA assay kit. SDS-PAGE and transfer was performed using NuPAGE Novex 4–12% sodium dodecyl sulfate-polyacrylamide gels and nitrocellulose membranes according to Invitrogen’s NuPAGE protocol. Membranes were blocked in PBS with 5% non-fat dry milk and 0.05% Tween-20 and incubated with the following primary antibodies and dilutions: anti-Loricrin (Covance) at 1:3000, anti-Caspase 14 (Novus Biological) at 1:3000, and anti-β-Actin-HRP (Sigma-Aldrich) at 1:30,000. Chemiluminescent detection was performed with ECL Plus detection reagent (Thermo Scientific Pierce) using the Fujifilm LAS-3000 imaging system. Densitometry measurements normalizing to β-Actin were made with the Fujifilm Mulitgauge version 2.0 software.
Calcium Green 5N (CG5N) and Fluorescein were purchased from Invitrogen (Carlsbad, CA).
Time domain FLIM measurements were performed using the same set up described in 21. Lifeteme data acquisition was controlled by the software SPCM (Becker and Hickl, Germany) which was also used to calculate the average lifetime decay times rendered in false color images in the central columns of figure 1A and B.
Phasor analysis of the lifetime data 34 was performed using the program SimFCS (Laboratory for Fluorescence Dynamics, UC-Irvine, CA) as described in 21. A fluorescein solution (pH 10) was used to reference the data, thus accounting for the instrument response function of the system.
The calcium concentration distribution at each layer of the sample (Figure 1) was calculated by binning the calcium range into four intervals: I1, concentrations lower than 1.5µM; I2, between 1.5 µM and 5 µM; I3, between 5 µM and 20 µM; I4, higher than 20µM. For each layer, the number of pixels with calcium concentration in each of the intervals was counted and normalized by the total number of pixels (frequency).
Data were analyzed using the paired, two-tailed Students t test, except in Figure 6, where they were analyzed using an ANOVA test.
This work was supported by National Institutes of Health grants AR051930 (T.M.) and AR19098 (P.E.) and the San Francisco Veterans Medical Center. The authors wish to thank Debra Crumrine for expert technical assistance.
Conflict of interest: The authors do not have any conflict of interest