the binding of il-6 to its receptor induces the activation of multiple signal transduction pathways such as jak / stats ( janus tyrosine kinase / signal transducers and activators of transcription ) pathway , ras / erk ( extracellular signal - regulated kinase ) pathway , and pi 3-k ( phosphotidylinositol 3-kinase)/akt pathway via gp130 tyrosine phosphorylation .
the roles of jak / stats pathway and ras / erk pathway in the biological effects of il-6 have been extensively investigated . however , what role pi 3-k / akt plays in il-6 signaling is less clear .
akt is a serine ( ser)/threonine ( thr ) protein kinase which resides within the cytosol in a catalytically inactive state in quiescent or serum - starved cells .
after stimulation of cells with growth factors and cytokines , akt is catalytically activated by phosphorylation at thr308 and ser473 . activated akt in turn phosphorylates downstream target molecules and induces the expression of anti - apoptosis proteins , which promote induction of its anti - apoptosis effect .
recently , growing evidence suggests the involvement of pi 3-k / akt in il-6-dependent survival and proliferative responses in several types of cells [ 3 - 6 ] .
still , whether pi 3-k / akt plays the same role in il-6-dependent growth of 7td1 mouse - mouse b cell hybridoma is not known . in our previous work , we showed that erk cascade but not stat3 contributed to il-6-dependent growth of 7td1 cells . here
we report that il-6 triggers activation of pi3-k / akt signaling in 7td1 cells , and that il-6-induced pi 3-k / akt activation is essential for the optimal growth of 7td1 cells .
-mercaptoethanol and recombinant human il-6 ( 10u / mg ) were added to the medium at final concentration of 5  10mol / l and 5 ng / ml , respectively .
wortmannin ( sigma ) was dissolved in dmso to 2.5 mm and stored at -20c .
dmso was added to control cells to keep concentrations of dmso ( < 0.1% ) equal in all samples .
the cells were pretreated with wortmannin for 30 min at 37c before il-6 was added to the medium .
the cells were seeded into 96-well plate and cultured in the presence of different dose of il-6 for 72 h. afterwards , 10 l mtt ( sigma , 5 mg / ml ) was added to each well , 4 h later , an equal volume of 10%sds-10 mm hcl was added to dissolve the blue crystals of formazan .
the samples were measured at od 570 nm by an elisa reader ( dynatech laboratories , inc . u.s.a . ) . after protein determination , total cell lysates ( 5  10cell / sample ) were boiled in 2  reducing sds loading buffer of equal volume for 10 min .
after electrophoresis , proteins were transferred to a 0.45 3 m pore - size nitrocellulose membrane at 40 v for 2 h. non - specific binding sites on the nitrocellulose membrane were blocked by incubation in blocking buffer ( 5% w / v , non - fat dried milk ) for 1 h at 37c .
the blots were washed once with tris - buffered saline ( 10 mm tris / hcl ph 7.5 , 150 mm nacl ) and incubated with the primary antibody for 1 h at 37c or overnight at 4c .
after the removal of excess primary antibody with three washes , the blots were incubated with a secondary antibody ( goat anti - mouse or goat anti - rabbit antibodies conjugated with horseradish peroxidase ) . the membrane was developed with enhanced chemiluminescence reagent and exposed to hyperfilm - ecl(amersham life science corp . ) for detection .
akt and phospho - akt ( ser473 ) antibodies were products of new england biolabs ( beverly , ma , u.s.a . ) .
anti-(human xiap ) antibody was a product of medical and biological laboratories ( naka , nagoya , japan ) .
anti - bcl-2 and anti - caspase-3 antibodies were obtained from santa cruz biotechnology ( santa cruz , ca , u.s.a . ) .
-mercaptoethanol and recombinant human il-6 ( 10u / mg ) were added to the medium at final concentration of 5  10mol / l and 5 ng / ml , respectively .
wortmannin ( sigma ) was dissolved in dmso to 2.5 mm and stored at -20c .
dmso was added to control cells to keep concentrations of dmso ( < 0.1% ) equal in all samples .
the cells were pretreated with wortmannin for 30 min at 37c before il-6 was added to the medium .
the cells were seeded into 96-well plate and cultured in the presence of different dose of il-6 for 72 h. afterwards , 10 l mtt ( sigma , 5 mg / ml ) was added to each well , 4 h later , an equal volume of 10%sds-10 mm hcl was added to dissolve the blue crystals of formazan .
the samples were measured at od 570 nm by an elisa reader ( dynatech laboratories , inc . u.s.a . ) .
after protein determination , total cell lysates ( 5  10cell / sample ) were boiled in 2  reducing sds loading buffer of equal volume for 10 min .
after electrophoresis , proteins were transferred to a 0.45 3 m pore - size nitrocellulose membrane at 40 v for 2 h. non - specific binding sites on the nitrocellulose membrane were blocked by incubation in blocking buffer ( 5% w / v , non - fat dried milk ) for 1 h at 37c .
the blots were washed once with tris - buffered saline ( 10 mm tris / hcl ph 7.5 , 150 mm nacl ) and incubated with the primary antibody for 1 h at 37c or overnight at 4c .
after the removal of excess primary antibody with three washes , the blots were incubated with a secondary antibody ( goat anti - mouse or goat anti - rabbit antibodies conjugated with horseradish peroxidase ) . the membrane was developed with enhanced chemiluminescence reagent and exposed to hyperfilm - ecl(amersham life science corp . ) for detection .
akt and phospho - akt ( ser473 ) antibodies were products of new england biolabs ( beverly , ma , u.s.a . ) .
anti-(human xiap ) antibody was a product of medical and biological laboratories ( naka , nagoya , japan ) .
anti - bcl-2 and anti - caspase-3 antibodies were obtained from santa cruz biotechnology ( santa cruz , ca , u.s.a . ) .
il-6 induced phosphorylation ( ser473 ) of akt , a downstream effector of pi 3-k , in 7td1 cells .
this activation by il-6 occurred as early as 5 min , and was dependent on the dose of il-6 .
il-6 stimulated phosphorylation of akt in a dose- and time - dependent manner in 7td1 cells .
7td1 cells were starved in il-6-free medium for 6 h. then the cells were cultured in the presence of different dose of il-6 for 5 min or in the presence of 1 ng / ml il-6 for 0 , 5 , 10 , 30 , 60 min respectively . after the cells were collected and washed , whole - cell extracts were prepared and subjected to western - blotting assay .
this figure is representative of 3 separate experiments . to investigate what role pi 3k / akt plays in the signal transduction of il-6 in 7td1 hybridoma cells
, we determined the effects of wortmannin , a pi 3-k specific inhibitor at 10100 nmol / l , on il-6-induced phosphorylation of akt and il-6-dependent growth of 7td1 cells .
it was found that wortmannin significantly antagonized il-6-induced phosphorylation of akt and il-6-dependent growth of 7td1 cells and the inhibitory effects of wortmannin were dependent on its concentration .
these data confirm that activation of akt is mediated by a pi 3-k - dependent mechanism and suggest that il-6-induced pi 3-k / akt activation is essential for the optimal growth of 7td1 cells ( fig .
effects of wortmannin on il-6-induced phosphorylation of akt ( a ) and il-6-dependent growth of 7td1 cells ( b ) .
7td1 cells were starved in il-6-free medium for 6 h. then the cells were pretreated with wortmannin or dmso of equal volume for 30 min at 37c before il-6 was added into the medium . (
after the cells were collected and washed , whole - cell extracts were prepared and subjected to western - blotting assay .
( b ) the cells were seeded into 96-well plate ( 2  10cells per well ) and cultured in the presence of different dose of il-6 for 72 h. afterwards , mtt assay was performed to determine the effect of wortmannin on il-6-dependent growth of 7td1 cells .
the preceding experiments suggest that il-6-induced pi 3-k / akt activation is essential for the optimal growth of 7td1 cells .
recent evidence has indicated that proteins of the inhibitor of apoptosis ( iap ) family , whose expression might be under the regulation of nf-b , can block apoptotic events by directly binding and inhibiting selected caspases .
a potent mammalian iap is x - linked iap ( xiap ) , for which the mechanism of action involves the direct binding and inhibition of caspase-3 and caspase-7 , two key effector proteases of apoptosis .
it is reported that the level of bcl-2 , but not bcl - xl and mcl-1 , decreased after il-6 deprivation . to examine the effects of il-6-induced pi3-k
/ akt activation on these apoptosis - related proteins , we further studied il-6-induced expression of bcl-2 as well as xiap and caspase-3 in 7td1 cells by western - blotting assay with bcl-2 , xiap , and caspase-3 antibodies , with or without wortmannin .
untreated 7td1 cells displayed significant levels of these apoptosis - related gene products ( fig .
3 ) . il-6 significantly up - regulated the levels of xiap and bcl-2 but had little effect on the level of caspase-3 .
. taken together , these data suggest that il-6 might induce up - regulation of xiap through pi 3-k / akt activation .
effects of il-6 and wortmannin on the expression of apoptosis - related proteins in 7td1 cells .
( a ) 7td1 cells were starved in il-6-free medium for 6 h. then the cells were cultured in the presence of 1 ng / ml il-6 for various periods as indicated . at the end of the incubation period , whole - cell extracts were prepared and subjected to western - blotting assay .
( b ) 7td1 cells were starved in il-6-free medium for 6 h. then the cells were pretreated with wortmannin or dmso of equal volume for 30 min at 37c before il-6 was added into the medium .
the cells were cultured for 1 h in the presence of il-6 . after the cells were collected and washed , whole - cell extracts were prepared and subjected to western - blotting assay .
il-6 induced phosphorylation ( ser473 ) of akt , a downstream effector of pi 3-k , in 7td1 cells .
this activation by il-6 occurred as early as 5 min , and was dependent on the dose of il-6 .
il-6 stimulated phosphorylation of akt in a dose- and time - dependent manner in 7td1 cells .
7td1 cells were starved in il-6-free medium for 6 h. then the cells were cultured in the presence of different dose of il-6 for 5 min or in the presence of 1 ng / ml il-6 for 0 , 5 , 10 , 30 , 60 min respectively . after the cells were collected and washed , whole - cell extracts were prepared and subjected to western - blotting assay .
to investigate what role pi 3k / akt plays in the signal transduction of il-6 in 7td1 hybridoma cells , we determined the effects of wortmannin , a pi 3-k specific inhibitor at 10100 nmol / l , on il-6-induced phosphorylation of akt and il-6-dependent growth of 7td1 cells .
it was found that wortmannin significantly antagonized il-6-induced phosphorylation of akt and il-6-dependent growth of 7td1 cells and the inhibitory effects of wortmannin were dependent on its concentration .
these data confirm that activation of akt is mediated by a pi 3-k - dependent mechanism and suggest that il-6-induced pi 3-k / akt activation is essential for the optimal growth of 7td1 cells ( fig .
effects of wortmannin on il-6-induced phosphorylation of akt ( a ) and il-6-dependent growth of 7td1 cells ( b ) .
7td1 cells were starved in il-6-free medium for 6 h. then the cells were pretreated with wortmannin or dmso of equal volume for 30 min at 37c before il-6 was added into the medium . (
after the cells were collected and washed , whole - cell extracts were prepared and subjected to western - blotting assay .
( b ) the cells were seeded into 96-well plate ( 2  10cells per well ) and cultured in the presence of different dose of il-6 for 72 h. afterwards , mtt assay was performed to determine the effect of wortmannin on il-6-dependent growth of 7td1 cells .
the preceding experiments suggest that il-6-induced pi 3-k / akt activation is essential for the optimal growth of 7td1 cells .
recent evidence has indicated that proteins of the inhibitor of apoptosis ( iap ) family , whose expression might be under the regulation of nf-b , can block apoptotic events by directly binding and inhibiting selected caspases .
a potent mammalian iap is x - linked iap ( xiap ) , for which the mechanism of action involves the direct binding and inhibition of caspase-3 and caspase-7 , two key effector proteases of apoptosis .
it is reported that the level of bcl-2 , but not bcl - xl and mcl-1 , decreased after il-6 deprivation . to examine the effects of il-6-induced pi3-k
/ akt activation on these apoptosis - related proteins , we further studied il-6-induced expression of bcl-2 as well as xiap and caspase-3 in 7td1 cells by western - blotting assay with bcl-2 , xiap , and caspase-3 antibodies , with or without wortmannin .
untreated 7td1 cells displayed significant levels of these apoptosis - related gene products ( fig .
3 ) . il-6 significantly up - regulated the levels of xiap and bcl-2 but had little effect on the level of caspase-3 .
. taken together , these data suggest that il-6 might induce up - regulation of xiap through pi 3-k / akt activation .
effects of il-6 and wortmannin on the expression of apoptosis - related proteins in 7td1 cells .
( a ) 7td1 cells were starved in il-6-free medium for 6 h. then the cells were cultured in the presence of 1 ng / ml il-6 for various periods as indicated . at the end of the incubation period ,
( b ) 7td1 cells were starved in il-6-free medium for 6 h. then the cells were pretreated with wortmannin or dmso of equal volume for 30 min at 37c before il-6 was added into the medium .
the cells were cultured for 1 h in the presence of il-6 . after the cells were collected and washed , whole - cell extracts were prepared and subjected to western - blotting assay .
il-6 inhibits physiological cell death and allows expansion of populations of serum - stimulated cells .
how il-6 can promote the growth of 7td1 cells remains elusive . in our previous work , we showed that erk cascade but not stat3 contributed to il-6-dependent growth of 7td1 cells .
however , activation of erk cascade seems not to be sufficient since mek inhibitor pd098059 pretreatment resulted in partial blockade of il-6-induced growth of 7td1 cells although il-6-induced activity of erk cascade can be completely blocked by pd098059 of the same concentration . in this work , we show that il-6-induced pi 3-k / akt activation is also essential for the optimal growth of 7td1 cells . taken together
, our data suggest that il-6 promotes the growth of 7td1 cells via activation of multiple signal transduction pathways including erk cascade and pi 3-k / akt pathway .
recently , growing evidence suggests the involvement of pi 3-k / akt in il-6-dependent survival and proliferative responses in several types of cells [ 3 - 6 ] .
our data are consistent with these findings , further confirming the important roles of pi 3-k / akt in il-6 signaling .
furthermore , we found that xiap , but not bcl-2 , might be a downstream target molecule of akt since both constitutive and il-6-induced expression of xiap , but not bcl-2 , in 7td1 cells was inhibited by wortmannin .
recent evidence has indicated that the expression of xiap might be under the regulation of nf-b .
pi 3-k / akt pathway is thought to be involved in the full activation of nf-b through phosphorylation of the rel proteins .
this is the first report that xiap is involved in il-6-mediated anti - apoptosis mechanism .
 backgroundrecently , growing evidence suggests the involvement of pi 3-k / akt in il-6-dependent survival and proliferative responses in several types of cells . however , whether pi 3-k / akt plays the same role in il-6-dependent growth of 7td1 mouse - mouse b cell hybridoma is not known.methodswe investigated the activation status of akt in 7td1 cells induced by il-6 . with pi 3-k specific inhibitor wortmannin 
 , we also investigated the biological roles of akt activation in 7td1 cells.resultsil-6 stimulated phosphorylation of akt in a dose- and time - dependent manner in 7td1 cells . 
 wortmannin significantly reduced il-6-induced phosphorylation of akt and il-6-dependent growth of 7td1 cells . 
 furthermore , wortmannin blocked il-6-induced up - regulation of xiap , but not bcl-2 in 7td1 cells.conclusionthe data suggest that il-6-induced pi 3-k / akt activation is essential for the optimal growth of 7td1 cells through up - regulation of anti - apoptosis proteins such as xiap . 