Vol. 5, 1205-1213, November 1994 Cell Growth & Differentiation 1205 Okadaic Acid-elicited Transcriptional Activation of Collagenase Gene Expression in HT-1 080 Fibrosarcoma Cells Is Mediated by JunB1 Jukka Westermarck, Jouko Lohi, Jorma Keski-Oja, and Veli-Matti Kah#{228}ri2 Departments of Dermatology (V-M. K.], Medical Biochemistry Ii. WI, and MediCity Research Laboratory U. W., V-M. K.1, University of Turku, FIN-20520 Turku, and Departments of Virology U. L., J. K-O.J and Dermatology U. K-O.J, University of Helsinki, FIN-00250 Helsinki, Finland Abstract Okadaic acid (OA) is a novel, non-phorbol ester-type tumor promoter, which is a specific inhibitor of protein phosphatases 1 and 2A. Treatment of human fibrosarcoma HT-1 080 cells with OA resulted in induction of collagenase and stromelysin-1 mRNA levels, while mRNA levels for tissue inhibitor of metalloproteinases-1 were enhanced to a lesser extent. Induction of collagenase and stromelysin-1 mRNA levels was dependent on protein synthesis. Exposure of HT- 1 080 cells to OA resulted in marked and persistent induction of junB, junD, and c-fos mRNA levels up to 24 h, while c-jun mRNA levels were only slightly elevated. In transiently transfected HT-1080 cells, OA-elicited activation of a 3.8-kilobase collagenase promoter/reporter gene construct was entirely dependent on junB expression, as determined by cotransfection with a junB antisense expression construct. Overexpression of JunB in HT-1 080 cells enhanced collagenase promoter activity by 10-fold, and OA augmented trans-activation of collagenase promoter by c-Jun and JunB. The results indicate that induction of collagenase gene expression by OA is mediated by enhanced JunB expression, as well as enhanced trans-activating capacity of AP-1 complexes containing c-Jun and JunB. These results also suggest that selective overexpression of junB may enhance invasive and metastatic potential of neoplastic cells. Introduction Degradation of extracellular matrix macromolecules is an important feature of tissue remodeling during embryonal development, angiogenesis, and tissue repair. Enhanced breakdown of connective tissue also plays an important role in various pathological conditions, including inflammatory disorders, e.g., rheumatoid arthritis and periodontitis (1-3). Degradation of basement membrane and extracellular ma- trix components are also essential steps in tumor cell inva- sion and metastasis (4). A major group of proteolytic en- zymes responsible for degradation of extracellular matrix is MMPs3 (matrixins; Refs. 1-3). An abundant component of the extracellular matrix of different connective tissues is collagen. Breakdown of fibril- lar collagens of types I, II, and III is initiated by interstitial collagenase (MMP-i) and continued by other matrixins in- cluding stromelysins and gelatinases (1-3). Latent interstitial collagenase is proteolytically activated in the extracellular space, and the maximal activation is achieved in the pres- ence of stromelysin-i (MMP-3), which also degrades base- ment membranes and gelatin (5). Thus, coordinate expression of interstitial collagenase and stromelysin is important for in- vasive and metastatic capacity of neoplastic cells. The activity of both interstitial collagenase and stromelysin-i is regulated at the tissue level by lIMP-i , which inactivates these enzymes by forming a stoichiometric complex with them (1-3). Expression of interstitial collagenase and stromelysin-i is induced by a variety of factors, including phorbol esters, epidermal growth factor, and cytokines TNF-a, IL-i , and leukoregulin (6-i 1). On the other hand, induction of both collagenase and stromelysin-i genes is potently inhibited by glucocorticoids and transforming growth factor-n (12- 1 5). Induction of both these genes by phorbol esters, IL-i, and TNF-a is preceded by a rapid and transient increase in c-jun and c-fos mRNA levels, which results in subsequent binding of c-Jun!c-Fos dimers to the AP-i elements in the promoter regions of the two genes (7, 8, 1 3, 1 6, 1 7). The role of JunB and JunD as mediators of the growth factor- elicited activation of collagenase and stromelysin-i gene expression has not been established. DNA binding affinity as well as trans-activating capacity of transcription factors, including c-Jun and c-Fos, is po- tently regulated by phosphorylation as a result of a balance between the activity of protein kinases and cellular protein phosphatases (reviewed in Refs. 18 and 1 9). We have re- cently shown that enhancement of cytokine and phorbol ester-elicited induction of collagenase gene expression in dermal fibroblasts by cyclosporin-A, an inhibitor of PP2B, is associated with persistent induction of c-jun expression (20). OA is a novel non-phorbol ester-type tumor promoter, which is a potent and selective inhibitor of serine!threo- nine-specific PP1 and PP2A (21 , 22). Thus, OA can be used to study the role oftranscription factor phoshorylation in the regulation of gene expression independently of activation of protein kinase A or C. It has been shown previously that OA enhances collagenase gene expression in lung adenocarci- Received 6/24/94; accepted 8/29/94. 1 This study was financially supported by grants from the Paulo Foundation, Turku University Foundation, Sigrid Jus#{233}lius Foundation, and the Academy of Finland. 2 To whom requests for reprints should be addressed, at Department of Derma- tology, University of Turku, Kiinamyllynkatu 4-8, FIN-20520 Turku, Finland. 3 The abbreviations used are: MMP, matrix metalloproteinase; TNF-a, tumor necrosis factor-a; IL-i , interleukin 1 ; PP1 , PP2A, PP2B, protein phosphatase 1 , 2A, or 2B; OA, okadaic acid; TIMP, tissue inhibitor of metalloproteinases; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydroge- nase; TPA, 12-O-tetradecanoylphorbol-13-acetate; kb, kilobase(s); CAT, chloramphenicol acetyl transferase; tk, thymidine kinase; bp, base pair(s); CRE, cAMP responsive element; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.