Up-Regulation of Transient Receptor Potential Canonical 1 (TRPC1) following Sarco(endo)plasmic Reticulum Ca²⁺ATPase 2 Gene Silencing Promotes Cell Survival: A Potential Role for TRPC1 in Darier's Disease

Abstract

The mechanism(s) involved in regulation of store operated calcium entry in Darier's disease (DD) is not known. We investigated the distribution and function of transient receptor potential canonical (TRPC) in epidermal skin cells. DD patients demonstrated up-regulation of TRPC1, but not TRPC3, in the squamous layers. Ca 2؉ influx was significantly higher in keratinocytes obtained from DD patients and showed enhanced proliferation compared with normal keratinocytes. Similar up-regulation of TRPC1 was also detected in epidermal layers of SERCA2 ؉/؊ mice. HaCaT cells expressed TRPC1 in the plasma membrane. Expression of sarco(endo)plasmic reticulum Ca 2؉ ATPase (SERCA)2 small interfering RNA (siRNA) in HaCaT cells increased TRPC1 levels and thapsigargin-stimulated Ca 2؉ influx, which was blocked by store-operated calcium entry inhibitors. Thapsigargin-stimulated intracellular Ca 2؉ release was decreased in DD cells. DD keratinocytes exhibited increased cell survival upon thapsigargin treatment. Alternatively, overexpression of TRPC1 or SERCA2-siRNA in HaCaT cells demonstrated resistance to thapsigargin-induced apoptosis. These effects were dependent on external Ca 2؉ and activation of nuclear factor-B. Isotretinoin reduced Ca 2؉ entry in HaCaT cells and decreased survival of HaCaT and DD keratinocytes. These findings put forward a novel consequence of compromised SERCA2 function in DD wherein up-regulation of TRPC1 augments cell proliferation and restrict apoptosis. We suggest that the anti-apoptotic effect of TRPC1 could potentially contribute to abnormal keratosis in DD. INTRODUCTIONDarier's disease (DD) is an autosomal dominant inherited skin disease, which is characterized by the loss of adhesion between epidermal cells and abnormal keratinization (Burge and Wilkinson 1992). Typical histological findings include focal areas of separation between suprabasal epidermal cells, unusual dyskeratosis with round dyskeratotic keratinocytes (Munro, 1992). Mutations in the sarco(endo)plasmic reticulum Ca 2ϩ ATPase (SERCA) isoform 2b (SERCA2b) gene leads to a loss of function, and these mutations have been shown to cause DD (Sakuntabhai et al., 1999). Most physiological systems are able to compensate for the loss of function of SERCA2b, possibly because of expression of other SERCA isoforms within those tissues (Dhitavat et al., 2003;Tavadia et al., 2004). However, of the multiple isoforms of SERCA1, -2, or -3, only SERCA2b is expressed in keratinocytes (Lytton and MacLennan, 1988;Ruiz-Perez et al., 1999). The mutated SERCA2 fails to sequester cytosolic Ca 2ϩ into the endoplasmic reticulum (ER) lumen, thereby disturbing the otherwise normal Ca 2ϩ homeostasis circuitry within the cells (Zhao et al., 2001;Ahn et al., 2003). Although oral retinoids, such as isotretinoin, have been shown to reduce hyperkeratosis (Burge and Wilkinson, 1992), the mechanism behind dysfunction of SERCA2 resulting in keratosis remains elusive.Ca 2ϩ is an integral signaling element that regulates numerous cellular processes (Clapham, 1995;Berridge e...

Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1

“…Functional consequences of Cav1 and STIM1 on SOCE were further examined by measuring NF-Bdriven luciferase reporter activity, NF-B (p65 subunit) nuclear translocation, and cell proliferation, both of which are regulated by SOCE (29,30). Store depletion promoted nuclear translocation of p65 subunit of NF-B, which was enhanced by TRPC1 expression, but was inhibited by La 3ϩ (Fig.…”

Darier’s disease: a calcium-signaling perspective

“…Predominantly in immune cells, ORAI1 constitutes the plasma membrane component of SOCs, whereas STIM1 functions as the ER Ca 2+ sensor which has a rather ubiquitous role in facilitation of SOCE by activating ORAI1 and/or TRPC (TRP canonical) components of SOCs [37,[39][40][41]. Although, the molecular identity of SOCs in keratinocytes remains elusive, increasing evidence suggests a profound involvement of TRPC channels in the skin system [42][43][44]. Elevating the cytosolic levels of Ca 2+ via the activation of plasma membrane SOCs not only aids replenishment of ER stores but also maintains cellular functions.…”

“…Darier's disease -TRPC1 perspective TRPC1, which is typically activated by ER store depletion, has been reported to be a critical component of SOCs in many cell types including endothelial, neuronal, smooth muscle and salivary gland cells, platelets, and keratinocytes [30,35,36,38,44,45]. Its interactions with the newly identified SOCE components, STIM1 and ORAI1, as a dynamic complex further amplify the physiological significance of TRPC1 as a SOC [41,46,47].…”

TRP Channels and Pruritus

“…Furthermore, DD keratinocytes show enhanced proliferation and apoptosis resistance, suggesting that TRPC1 is involved in the abnormal keratinization in DD epidermis. Importantly, experiments performed on SERCA2b KO mice as well as on human epidermal HaCaT keratinocytes, in which expression of SERCA2b was silenced by siRNA, concluded similar results [368]. Other studies also show that TRPC1/TRPC4 channels were important for keratinocyte differentiation as siRNA based silencing of these channels prevented the induction of Ca 2+ -induced differentiation.…”

“…This malformation causes a severe differentiation disorder of keratinocytes and is very often associated with intense pruritus [367,368]. TRPC1-mediated Ca 2+ influx was significantly higher in keratinocytes obtained from DD patients.…”