Ee principal, ubiquitously expressed ER stress sensors; PKR-like ER kinase (PERK

Ee principal, ubiquitously expressed ER stress sensors; PKR-like ER kinase (PERK), inositol-requiring enzyme 1a (IRE1a) and activating transcription factor 6 (ATF6) mediate the UPR [8,9]. Once activated these proteins transduce signals that lead to a transient inhibition in protein translation and transcriptional increases of ER chaperones and degradation components in an attempt to increase protein folding and eliminate misfolded proteins. In addition to the three main ER stress sensors, additional proteins related to ATF6 such as Old Astrocyte SpecificallyInduced Substance (OASIS) (also named CREB3L1) are expressed in certain cell types [10,11,12]. Similar to ATF6, OASIS is a type II membrane protein with a cytoplasmic N-terminal transcription factor domain and an ER luminal C-terminal domain. OASIS mRNA was first found to be induced in long-term cultured astrocytes and in response to cryo-injury in the mouse cerebral cortex [13]. Subsequent studies found that OASIS mRNA is expressed in a variety of human tissues with predominant expression in pancreas and prostate [14]. More recent studies have shown that OASIS may have a role in differentiation and development of odontoblasts, osteoblasts and pancreatic b-cells [15,16,17,18,19]. Imaizumi and colleagues were the first to identify that OASIS is an ER stress transducer that translocates from the ER to the Golgi upon ER stress, where it is cleaved by regulated intramembrane proteolysis to release a cytosolic fragment that translocates to nucleus 1662274 to bind CRE and ERSE (ER stress responsive element) DNA elements [20,21]. OASIS overexpressed in rat astrocytes up-regulates the expression of GRP78 NT 157 manufacturer chaperone, indicating that it may contribute to induction of the UPR [20]. However, OASIS induces the expression of other genes such as extracellular matrix components rather than typicalOASIS in Human Glioma CellsER stress response genes in osteoblasts [16] and pancreatic b-cells [18]. ER stress has been shown to occur in cancer cells potentially due to the hypoxic conditions experienced by cancer cells in vivo [22] and the ER stress response has been suggested to be a potential pathway that can be pharmacologically exploited to induce apoptosis in gliomas [23]. The extracellular matrix has been implicated in cancer cell metastases [24]. For example, in glioblastoma multiforme, the most common brain cancers that are also particularly aggressive [25], the extracellular matrix is involved in cell invasion and migration [26,27]. Given that OASIS is induced by ER stress and may modulate the extracellular matrix we examined OASIS expression in several human glioma cell lines and the role of this protein in the ER stress response, extracellular matrix production and cell migration.100 nM siRNA using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.Wound Healing AssayTo monitor migration rate, U373 cells (0.46106) were transfected with 100 nM control or OASIS siRNA for 3 days and incubated at 37uC until cells reached 90 confluence to form a monolayer in a 6 well plate. A p200 pipette tip was used to create a uniform scratch of the cell monolayer followed by a wash with PBS. Fresh DMEM medium (25 mM glucose, 2 mM L-glutamine, 10 FBS, 100 U/ml Acetovanillone penicillin, 100mg/ml streptomycin) was added and the cells were incubated for 24?8 h. Representative DIC images of wound healing were monitored with Olympus fluorescence inverted microscope (IX71). Wound closure was determined.Ee principal, ubiquitously expressed ER stress sensors; PKR-like ER kinase (PERK), inositol-requiring enzyme 1a (IRE1a) and activating transcription factor 6 (ATF6) mediate the UPR [8,9]. Once activated these proteins transduce signals that lead to a transient inhibition in protein translation and transcriptional increases of ER chaperones and degradation components in an attempt to increase protein folding and eliminate misfolded proteins. In addition to the three main ER stress sensors, additional proteins related to ATF6 such as Old Astrocyte SpecificallyInduced Substance (OASIS) (also named CREB3L1) are expressed in certain cell types [10,11,12]. Similar to ATF6, OASIS is a type II membrane protein with a cytoplasmic N-terminal transcription factor domain and an ER luminal C-terminal domain. OASIS mRNA was first found to be induced in long-term cultured astrocytes and in response to cryo-injury in the mouse cerebral cortex [13]. Subsequent studies found that OASIS mRNA is expressed in a variety of human tissues with predominant expression in pancreas and prostate [14]. More recent studies have shown that OASIS may have a role in differentiation and development of odontoblasts, osteoblasts and pancreatic b-cells [15,16,17,18,19]. Imaizumi and colleagues were the first to identify that OASIS is an ER stress transducer that translocates from the ER to the Golgi upon ER stress, where it is cleaved by regulated intramembrane proteolysis to release a cytosolic fragment that translocates to nucleus 1662274 to bind CRE and ERSE (ER stress responsive element) DNA elements [20,21]. OASIS overexpressed in rat astrocytes up-regulates the expression of GRP78 chaperone, indicating that it may contribute to induction of the UPR [20]. However, OASIS induces the expression of other genes such as extracellular matrix components rather than typicalOASIS in Human Glioma CellsER stress response genes in osteoblasts [16] and pancreatic b-cells [18]. ER stress has been shown to occur in cancer cells potentially due to the hypoxic conditions experienced by cancer cells in vivo [22] and the ER stress response has been suggested to be a potential pathway that can be pharmacologically exploited to induce apoptosis in gliomas [23]. The extracellular matrix has been implicated in cancer cell metastases [24]. For example, in glioblastoma multiforme, the most common brain cancers that are also particularly aggressive [25], the extracellular matrix is involved in cell invasion and migration [26,27]. Given that OASIS is induced by ER stress and may modulate the extracellular matrix we examined OASIS expression in several human glioma cell lines and the role of this protein in the ER stress response, extracellular matrix production and cell migration.100 nM siRNA using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.Wound Healing AssayTo monitor migration rate, U373 cells (0.46106) were transfected with 100 nM control or OASIS siRNA for 3 days and incubated at 37uC until cells reached 90 confluence to form a monolayer in a 6 well plate. A p200 pipette tip was used to create a uniform scratch of the cell monolayer followed by a wash with PBS. Fresh DMEM medium (25 mM glucose, 2 mM L-glutamine, 10 FBS, 100 U/ml penicillin, 100mg/ml streptomycin) was added and the cells were incubated for 24?8 h. Representative DIC images of wound healing were monitored with Olympus fluorescence inverted microscope (IX71). Wound closure was determined.