Human keratinocyte cell lines differ in the expression of the collagenolytic matrix metalloproteinases-1,-8, and -13 and of TIMP-1.
We investigated cells and conditioned media of the three human keratinocyte cell lines HaCaT (non-tumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic) with regard to production and secretion of the collagenases-1 to -3 (MMP-1, MMP-8 and MMP-13) and TIMP-1 using semi-nested RT-PCR, Western blots, ELISA, immunocytochemistry and casein zymography. Transcripts of MMP-1, -8, -13 and TIMP-1 were detected in all cell lines by RT-PCR and the corresponding proteins were found in the cytoplasm of all three cell lines by Western blot analysis and/or immunocytochemistry. The conditioned media of the malignant II-4RT cells contain significantly more MMP-1 and MMP-8 than those of HaCaT or A5 as evidenced by immunoblotting and ELISA. In addition to the presence of latent MMP-1, zymography also detected the active form of this enzyme. TIMP-1 was found only in extracts of all three cell lines, predominantly in A5. This study clearly indicates that the epithelial tumor cells synthesize different collagenases and TIMP-1. The malignant clone secretes increased amounts of distinct collagenases compared to the non-tumorigenic cell line, thereby verifying a correlation between biological behaviour and the amount of collagenases. In addition, we provide clear evidence that MMP-8 is not exclusively found in polymorphonuclear granulocytes, but also in keratinocyte cell lines.
Department of Clinical Chemistry and Clinical Biochemistry, University Hospital of Surgery, Ludwig-Maximilians-University, Munich, Germany.
B E
BE
Bachmeier
A G
AG
Nerlich
P
P
Boukamp
R
R
Lichtinghagen
H
H
Tschesche
H
H
Fritz
E
E
Fink
D001483:Base Sequence; D015153:Blotting, Western; D002457:Cell Extracts; D002460:Cell Line; D003094:Collagen / Q000378:metabolism*; D017077:Culture Media, Conditioned; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000201:enzymology* / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*
D002457:Cell Extracts; D017077:Culture Media, Conditioned; D017931:DNA Primers; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D003094:Collagen; D020782:Matrix Metalloproteinases