Suppression of membrane-type 1 matrix metalloproteinase (MMP)-mediated MMP-2 activation and tumor invasion by testican 3 and its splicing variant gene product, N-Tes.
Using expression cloning to screen a human fetal kidney cDNA library for regulator(s) of pro-matrix metalloproteinase (MMP)-2 processing mediated by membrane-type (MT) 1 MMP, we isolated a cDNA whose product interfered with pro-MMP-2 activation. It encodes the NH(2)-terminal 313-amino acid region of a calcium-binding proteoglycan, testican 3, with a 3-amino acid substitution at the COOH terminus and thus was named N-Tes. N-Tes comprises a signal peptide, a unique domain, a follistatin-like domain, and a Ca(2+)-binding domain but lacks a COOH-terminal thyroglobulin domain and two putative glycosaminoglycan attachment sites of testican 3. Pro-MMP-2 activation by MT3-MMP was also inhibited by the coexpression of N-Tes. Immunoprecipitation analysis demonstrated direct interaction of N-Tes with either MT1-MMP or MT3-MMP. Expression of testican 1 or testican 3 but not testican 2 also inhibited pro-MMP-2 activation by either MT1-MMP or MT3-MMP. Deletion and substitution of amino acids residues in N-Tes revealed that the unique NH(2)-terminal domain of N-Tes is responsible for the inhibition of pro-MMP-2 activation by MT-MMPs. Expression of N-Tes and testican 3 was detected in normal brain but down-regulated in glioma tissues. Transfection of either the N-Tes or testican 3 gene into U251 glioma cells or Madin-Darby canine kidney cells transformed by erbB2 suppressed their invasive growth in collagen gel. These results suggest that both N-Tes and testican 3 would interfere with tumor invasion by inhibiting MT-MMPs.
Nakada
M
M
Department of Molecular Virology and Oncology, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-0934, Japan.
Yamada
A
A
Takino
T
T
Miyamori
H
H
Takahashi
T
T
Yamashita
J
J
Sato
H
H
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D015536:Down-Regulation; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018093:Gelatinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015723:Gene Library; D005910:Glioma / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000502:physiology; D053513:Matrix Metalloproteinase 16; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D020033:Protein Isoforms; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004792:Enzyme Precursors; C507102:MMP16 protein, human; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D011509:Proteoglycans; D012333:RNA, Messenger; C504369:SPOCK1 protein, human; C504404:SPOCK2 protein, human; C504409:SPOCK3 protein, human; D018093:Gelatinases; D053513:Matrix Metalloproteinase 16; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase