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
. 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.