Erythropoietin suppresses epithelial to mesenchymal transition and intercepts Smad signal transduction through a MEK-dependent mechanism in pig kidney (LLC-PK1) cell lines.
OBJECTIVE
Tumor growth factor-beta1 (TGF-beta1) plays a pivotal role in processes like kidney epithelial-mesenchymal transition (EMT) and interstitial fibrosis, which correlate well with progression of renal disease. Little is known about underlying mechanisms that regulate EMT. Based on the anatomical relationship between erythropoietin (EPO)-producing interstitial fibroblasts and adjacent tubular cells, we investigated the role of EPO in TGF-beta1-mediated EMT and fibrosis in kidney injury.


METHODS
We examined apoptosis and EMT in TGF-beta1-treated LLC-PK1 cells in the presence or absence of EPO. We examined the effect of EPO on TGF-beta1-mediated Smad signaling. Apoptosis and cell proliferation were assessed with flow cytometry and hemocytometry. We used Western blotting and indirect immunofluorescence to evaluate expression levels of TGF-beta1 signal pathway proteins and EMT markers.


RESULTS
We demonstrated that ZVAD-FMK (a caspase inhibitor) inhibited TGF-beta1-induced apoptosis but did not inhibit EMT. In contrast, EPO reversed TGF-beta1-mediated apoptosis and also partially inhibited TGF-beta1-mediated EMT. We showed that EPO treatment suppressed TGF-beta1-mediated signaling by inhibiting the phosphorylation and nuclear translocation of Smad 3. Inhibition of mitogen-activated protein kinase kinase 1 (MEK 1) either directly with PD98059 or with MEK 1 siRNA resulted in inhibition of EPO-mediated suppression of EMT and Smad signal transduction in TGF-beta1-treated cells.


CONCLUSIONS
EPO inhibited apoptosis and EMT in TGF-beta1-treated LLC-PK1 cells. This effect of EPO was partially mediated by a mitogen-activated protein kinase-dependent inhibition of Smad signal transduction.
Chen
Chien-Liang
CL
Division of Nephrology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Chou
Kang-Ju
KJ
Lee
Po-Tsang
PT
Chen
Ying-Shou
YS
Chang
Tsu-Yuan
TY
Hsu
Chih-Yang
CY
Huang
Wei-Chieh
WC
Chung
Hsiao-Min
HM
Fang
Hua-Chang
HC
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004848:Epithelium / Q000187:drug effects* / Q000502:physiology; D004921:Erythropoietin / Q000494:pharmacology*; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D008648:Mesoderm / Q000187:drug effects* / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D013552:Swine; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004921:Erythropoietin; D020929:Mitogen-Activated Protein Kinase Kinases; D020169:Caspases