Aims: The cross-talk between TGF-β/Smad and MAPK signaling is known to be involved in hepatocellular carcinoma (HCC) development.The phosphorylation and subcellular trafficking of Smads is the key platform in the interaction between MAPKs and TGF-β1/Smad signaling pathway.PAI-1 is the downstream of TGF-β/Smad signal pathway.Previous studies showed that MAPK specific inhibitors, including ERK inhibitor (PD98059), JNK inhibitor (SP600125) or p38 inhibitor (SB203580) could inhibit the transcription of PAI-1 in HepG2 cells.Different from JNK or p38 inhibitor, ERK inhibitor had little effect on the phosphorylation of Smad2/3 and the formation of Smad2/3/4 complexes.We hypothesize that MAPK inhibitors, especially ERK inhibitor, play a role in regulating the transport of Smads and we conducted this study to test this hypothesis and explore the potential mechanism preliminarily.Methods: After starving overnight in serum-free medium, HepG2 cells were incubated in the absence or presence of ERK inhibitor, JNK inhibitor or p38 inhibitor for 5 h, respectively; subsequently stimulated with TGF-β1 for 1 h.We detected the intracellular location of Smads (pSmad2C, pSmad2L, pSmad3C, pSmad3L and Smad4) and Imp7 or 8 (responsible for Smads translocation into nucleus) by immunofluorescence study and investigated the expression of Imp7 or 8 by immuneblot analysis.Results: pSmad2C, pSmad2L, pSmad3C and pSmad3L were increased and translocated into nucleus under TGF-β1 treatment.The presence of ERK inhibitor only inhibited the phosphorylation of Smad2C and had no significant influence on the phosphorylation of other three phosphorylated Smads, but affected the nuclear accumulation of pSmad2C, pSmad2L, pSmad3C and pSmad3L.JNK inhibitor inhibited the phosphorylation of Smad2C, Smad2L or Smad3L and affected their nuclear accumulation.p38 inhibitor also inhibited the phosphorylation of Smad2C, Smad2L or Smad3L and affected the nuclear accumulation of pSmad2C or pSmad2L.All three MAPK inhibitors attenuated the nuclear distribution of Smad4.Three MAPK inhibitors attenuated the expression and nuclear accumulation of Imp7 stimulated by TGF-β1, while ERK and JNK inhibitors attenuated the expression and nuclear accumulation of Imp8.Discussion: PAI-1 induction and tumor cell invasion required both pSmad2L (Ser-245/250/255)/C and pSmad3L (Ser-213) activity.In HepG2 cells, our previous study have found that TGF-β1 induced the expression of pSmad2C, pSmad2L and pSmad3L, increased PAI-1 expression and promoted the capacity of invasion.All three MAPK inhibitors suppressed PAI-1 expression.Among them, JNK or p38 inhibitor inhibited the expression of pSmad2C, pSmad2L and pSmad3L, but ERK inhibitor only inhibited the expression of pSmad2L, and did not affect the expression of pSmad2C, pSmad3L.Immunofluorescence measure in this study showed that the nuclear distribution of pSmad2C, pSmad3L decreased after treating by PD98059, although the expression of pSmad2C, pSmad3L remained unchanged, suggesting that ERK inhibitor may inhibit PAI-1 expression via regulating the transport of phosphorylated R-Smads.The invasion induction effect ofpSmad2C/L and pSmad3L need Smad4 and Smad2/3/4 complex formation.Intracellular distribution of Smad4 also reflects the distribution of phosphorylated R-Smads or R-Smads-Smad4 complexes.In our study, after treating by JNK or p38 inhibitor, the formation of Smad2/3/4 complexes was blocked because phosphorylated R-Smads were inhibited, subsequently the nuclear accumulation of Smad4 decreased.Although ERK inhibitor did not inhibited the Smad2/3/4 complexes, it reduced the nuclear distribution of Smad4, further proving the effect of ERK inhibitor on the translocation of Smad2/3/4 complexes.It has been reported that the subcellular distribution of representative cargo proteins is similar to that of importin β.We observed that the nuclear accumulation of Imp7/8 was impaired by MAPK inhibitors, similar to that of Smads, suggesting MAPK inhibitors may regulate the Smads import by affecting Imp7 or 8.Previous studies have reported that knockdown of Imp7 and Imp8 strongly inhibited TGF-β induced Smad2/3 nuclear translocation, while overexpressing Imp8 could increase the concentration of Smad3 or 4 in nucleus.The expression level of imp7 or imp8 directly affects the Smads nuclear translocation.Our data showed that ERK inhibitor decreased the expression of imp7 or imp8, besides JNK or p38 inhibitor.We proposed that inhibiting imp7 or imp8 is an important mechanism in regulating Smads translocation by MAPK inhibitors.In summary, the study has demonstrated that MAPK inhibitors, especially ERK inhibitor, regulate TGF-β1/Smad signaling pathway by reducing Smads nuclear accumulation.Inhibiting imp7 or imp8 is an important mechanism in regulating Smad translocation by MAPK inhibitors.