MicrogliaActivation-Induced Cell Death of Brain Gene-1 as an Apoptotic Sensitizer in Role of Antiproliferative B Cell Translocation

Mouse brain microglial cells undergo apoptosis on exposure to inﬂammatory stimuli, which is considered as an autoregulatory mechanism to control their own activation. Here, we present evidence that an antiproliferative B cell translocation gene 1 ( BTG1 ) constitutes a novel apoptotic pathway of LPS/IFN- (cid:1) -activated microglia. The expression of BTG1 was synergistically enhanced by LPS and IFN- (cid:1) in BV-2 mouse microglial cells as well as in primary microglia cultures. Levels of BTG1 expression inversely correlated with a proliferative capacity of the microglial cells. Tetracycline-based conditional expression of BTG1 not only suppressed microglial proliferation but also increased the sensitivity of microglial cells to NO-induced apoptosis, suggesting a novel mechanism of cooperation between LPS and IFN- (cid:1) in the induction of microglial apoptosis. An increase in BTG1 expression, however, did not affect microglial production of NO, TNF- (cid:2) , or IL-1 (cid:3) , indicating that the antiproliferative BTG1 is important in the activation-induced apoptosis of microglia, but not in the activation itself. The synergistic action of LPS and IFN- (cid:1) in the microglial BTG1 induction and apoptosis was dependent on the Janus kinase/STAT1 pathway, but not IFN-regulatory factor-1, as demonstrated by a pharmacological inhibitor of Janus kinase (AG490), STAT1 dominant negative mutant, and IFN-regulatory factor-1-deﬁcient mice. Taken together, antiproliferative BTG1 may participate in the activation-induced cell death of microglia by lowering the threshold for apoptosis; BTG1 increases the cells by a sequential addition of 4 M guanidinium thiocyanate, 2 M sodium acetate, and acid phenol-chloroform. Reverse transcription was conducted using Superscript (Life Technologies-BRL) and oligodeoxythymidylate primer. PCR ampliﬁcation using primer sets speciﬁc for IRF-1 was conducted at 60°C annealing temperature for 30 cycles. PCR for BTG1 and (cid:3) -actin was conducted at 55°C annealing temperature for 30 cycles. Nucleotide se- quences of the primers were based on published cDNA sequences of mouse BTG1 , (cid:3) -actin, and IRF-1 ( BTG1 forward, GAT TGG ACT GAG CAG TCA GGA; BTG-1 reverse, TCT TAA CCT GAT ACA GTC ATC; (cid:3) -actin forward, ATC CGT AAA GAC CTC TAT GC; (cid:3) -actin reverse, AAC GCA GCT CAG TAA CAG TC; IRF-1 forward, TCT GAG TGG ATG CAG ATG GAC; IRF-1 reverse, GGT CAG AGA CCC AAA CTA TGG TCG). The lengths of PCR product for each primer set were 261 bp for BTG1 , 287 bp for (cid:3) -actin, and 426 bp for IRF-1, respectively. For Northern blot analysis, isolated total RNA (20 (cid:4) g/sample) was electrophoresed in a 1.0% agarose gel containing 0.7% formaldehyde, 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA. RNA was transferred to the Nytran membrane (Schleicher & Schuell, Keene, NH). After UV cross- linking, the membranes were hybridized with a Bam HI fragment (511 bp) of BTG1 cDNA probe (10 6 cpm/ml). The membranes were then washed at 65°C, dried, and exposed to x-ray ﬁlms. and tetracycline. In a selected clone, BV-2/tet3, BTG1 induction was evaluated by removal of tetracycline from the growth medium. Western blot detection of BTG1 protein and densitometric analysis indicated (cid:7) 5 fold increase in BTG1 protein level in the cells grown in the absence of tetracycline ( (cid:1) tet) compared with the cells grown in the presence of tetracycline ( (cid:2) tet). BTG1 protein level was normalized to (cid:2) -tubulin. Results are representative of three independent experiments.