We addressed the requirement for stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca 2ϩ -sensor, and Orai1, a Ca 2ϩ selective channel, in regulating Ca 2ϩ entry through the store-operated channels mouse transient receptor potential canonical (TRPC) 4 or human TRPC1. Studies were made using murine and human lung endothelial cells (

(24,38). TRP genes encode a family of proteins with six transmembrane helices that are divided into seven subfamilies: TRPC (canonical or classical), TRPV (vaniloid related), TRPM (melastatin related), TRPA (ankyrin related), TRPML (mucolipin related), TRPP (polycystin related), and TRPN (no mechanoreceptor potential C) (22,30). Members of the TRPC subfamily contain 700 to 1,000 amino acids, and seven isoforms (TRPC1 to 7) are expressed in mammalian cells. Mammalian TRPCs are grouped into four subfamilies. One group consists of TRPC1, TRPC4, and TRPC5. Their activation is dependent on Ca 2ϩ store depletion and they have high Ca 2ϩ selectivity as assessed by their sensitivity to La 3ϩ (24). TRPC4 and TRPC5 are activated by G protein-coupled receptors and receptor tyrosine kinases coupled to phospholipase C. TRPC1 is closely related to TRPC4 and TRPC5; although it forms SOCs, it is a less selective Ca 2ϩ channel. TRPC3, TRPC6, and TRPC7 form store-independent nonselective cation channels activated by diacylglycerol (6); however, a store-dependent activation mechanism has been described for human TRPC3 (6). TRPC2 is believed to be a pseudogene in humans, and its function is unclear (22).We (25-27) and others (3) have shown that the TRPC1 isoform, prominently expressed in human vascular endothelial cells, is essential for SOCE. TRPC1 is localized within cholesterol-rich plasma membrane invaginations termed caveolae (17) that are coated with the 22-kDa protein caveolin-1 (Cav-1). Studies showed that Ca 2ϩ influx occurred in caveolar microdomains in response to Ca 2ϩ depletion of endoplasmic reticulum (ER) store in endothelial cells (10,11,13). Furthermore, studies have shown that the binding of Cav-1 with both the NH 2 and COOH termini of TRPC1 was necessary for the caveolar distribution of TRPC1 (2). Patel et al. (29)

Calcium signaling and calcium transport play a key role during osteoblast differentiation and bone formation. Here, we demonstrate that DMP1 mediated calcium signaling, and its downstream effectors play an essential role in the differentiation of preosteoblasts to fully functional osteoblasts. DMP1, a key regulatory bone matrix protein, can be endocytosed by preosteoblasts, triggering a rise in cytosolic levels of calcium that initiates a series of downstream events leading to cellular stress. These events include release of store-operated calcium that facilitates the activation of stress-induced p38 MAPK leading to osteoblast differentiation. However, chelation of intracellular calcium and inhibition of the p38 signaling pathway by specific pharmacological inhibitors and dominant negative plasmid suppressed this activation. Interestingly, activated p38 MAPK can translocate to the nucleus to phosphorylate transcription factors that coordinate the expression of downstream target genes such as Runx 2, a key modulator of osteoblast differentiation. These studies suggest a novel paradigm by which DMP1-mediated release of intracellular calcium activates p38 MAPK signaling cascade to regulate gene expression and osteoblast differentiation.Osteoblasts can react to a variety of biological signals. Among these, calcium signaling is essential for the proliferation and differentiation of osteoblasts. Earlier studies have shown that treating osteoblasts with parathyroid hormone or vitamin D 3 induces an increase in intracellular calcium ([Ca 2ϩ ] i ) by increasing the release of Ca 2ϩ from the intracellular stores (1-5). Store-operated Ca 2ϩ channels, which are activated in response to Ca 2ϩ store depletion, control homeostasis between the extracellular Ca 2ϩ reservoir and intracellular Ca 2ϩ storage and control a wide range of cellular functions.Dentin matrix protein 1 (DMP1) initially identified and localized in the mineralized dentin and bone matrix (6) is thought to play a regulatory role only in the calcification of the extracellular matrix. Apart from its role in mineralization, one of the putative functions of DMP1 is its involvement during differentiation of osteoblasts and odontoblasts (7-9). DMP1-null mice displayed severe defects in bone formation (10). We had shown earlier that DMP1 is specifically localized in the nucleus of differentiating osteoblasts and odontoblasts, and this translocation from the extracellular matrix is facilitated by the endocytic receptor GRP78 (11). The 78-kDa glucose-regulated protein (GRP78) is a calcium-binding molecular chaperone expressed in the endoplasmic reticulum of eukaryotic cells. Identification of GRP78 as a cell surface receptor for DMP1 is particularly interesting as its induction is a protective response against several kinds of stress, including ER 2 Ca 2ϩ depletion and accumulation of unglycosylated proteins (12, 13). However, the specific signaling pathways activated following DMP1 stimulus and osteoblast differentiation are not delineated yet.p38 MAPKs are widely e...

The Ca2+ sensor STIM1 is crucial for activation of store-operated Ca2+ entry (SOCE) through transient receptor potential canonical and Orai channels. STIM1 phosphorylation serves as an “off switch” for SOCE. However, the signaling pathway for STIM1 phosphorylation is unknown. Here, we show that SOCE activates AMP-activated protein kinase (AMPK); its effector p38β mitogen-activated protein kinase (p38β MAPK) phosphorylates STIM1, thus inhibiting SOCE in human lung microvascular endothelial cells. Activation of AMPK using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in STIM1 phosphorylation on serine residues and prevented protease-activated receptor-1 (PAR-1)-induced Ca2+ entry. Furthermore, AICAR pretreatment blocked PAR-1-induced increase in the permeability of mouse lung microvessels. Activation of SOCE with thrombin caused phosphorylation of isoform α1 but not α2 of the AMPK catalytic subunit. Moreover, knockdown of AMPKα1 augmented SOCE induced by thrombin. Interestingly, SB203580, a selective inhibitor of p38 MAPK, blocked STIM1 phosphorylation and led to sustained STIM1-puncta formation and Ca2+ entry. Of the three p38 MAPK isoforms expressed in endothelial cells, p38β knockdown prevented PAR-1-mediated STIM1 phosphorylation and potentiated SOCE. In addition, inhibition of the SOCE downstream target CaM kinase kinase β (CaMKKβ) or knockdown of AMPKα1 suppressed PAR-1-mediated phosphorylation of p38β and hence STIM1. Thus, our findings demonstrate that SOCE activates CaMKKβ-AMPKα1-p38β MAPK signaling to phosphorylate STIM1, thereby suppressing endothelial SOCE and permeability responses.

Dental-pulp tissue is often exposed to inflammatory injury. Sequested growth factors or angiogenic signaling proteins that are released following inflammatory injury play a pivotal role in the formation of reparative dentin. While limited or moderate angiogenesis may be helpful for dental pulp maintenance, the induction of significant level of angiogenesis is probably highly detrimental. Hitherto, several studies have addressed the effects of proinflammatory stimuli on the survival and differentiation of dental-pulp stem cells (DPSC), in vitro. However, the mechanisms communal to the inflammatory and angiogenic signaling involved in DPSC survival and differentiation remain unknown. Our studies observed that short-term exposure to TNF-α (6 and 12 hours [hrs]) induced apoptosis with an upregulation of VEGF expression and NF-κB signaling. However, long-term (chronic) exposure (14 days) to TNF-α resulted in an increased proliferation with a concomitant shortening of the telomere length. Interestingly, DPSC pretreated with Nemo binding domain (NBD) peptide (a cell permeable NF-κB inhibitor) significantly ameliorated TNF-α- and/or VEGF-induced proliferation and the shortening of telomere length. NBD peptide pretreatment significantly improved TNF-α-induced downregulation of proteins essential for differentiation, such as bone morphogenic proteins (BMP)-1 & 2, BMP receptor isoforms-1&2, trasnforming growth factor (TGF), osteoactivin and osteocalcin. Additionally, inhibition of NF-κB signaling markedly increased the mineralization potential, a process abrogated by chronic exposure to TNF-α. Thus, our studies demonstrated that chronic inflammation mediates telomere shortening via NF-κB signaling in human DPSC. Resultant chromosomal instability leads to an emergence of increased proliferation of DPSC, while negatively regulating the differentiation of DPSC, in vitro.