Received 6/1 5/92. t This work was supported by a grant from the American Institute for Cancer Research (K. E. C., I. P.), NIH Biomedical Research Support Grant Award 507 RR07160 (K. E. C.), and Grant R29CA48990 from the NIH (L. P.). 0. 0. was supported by a fellowship from Johnson & Johnson. K. E. C. is the recipient of a Junior Faculty Research Award from the American Cancer Society. This work was presented in preliminary form at the annual meeting of the American Association for Cancer Research, Washington DC, May 23-26, 1990, and the annual meeting of the American Society for Biochemistry and Molecular Biology, New Orleans, LA, June 4-7, 1990. 2 To whom requests for reprints should be addressed, at Department of Chemistry and Biochemistry, Physical Sciences Building, University of South Carolina, Columbia, SC 29208. 3 The abbreviations used are: RA, retinoic acid; RARs, retinoic acid receptors; TGF-fl, transforming growth factor fi; HKc, human keratino- cytes, HKc/HPV16, immortalized human keratinocyte lines obtained by transfection of normal human keratinocytes with human papillomavirus type 16 DNA; HKc/GFI, a growth factor-independent human keratinocyte line; BPE, bovine pituitary extract; EGF, epidermal growth factor; BSA, bovine serum albumin; HEPES, 4-(2-hydroxyethyl)-1 -piperazineethane- sulfonic acid; SELISAs, sandwich enzyme-linked immunosorbent assays; SSC, standard saline citrate; pRB, product of the retinoblastoma suscep- tibility gene; 505, sodium dodecyl sulfate; cDNA, complementary DNA; kb, kilobase(s). Vol. 3, 763-772, November 1992 Cell Growth & Differentiation 763 Retinoic Acid Induces Secretion of Latent Transforming Growth Factor $ and $2 in Normal and Human Papillomavirus Type 1 6-immortalized Human Keratinocytes1 Ayse Batova, David Danielpour, Lucia Pirisi, and Kim E. Creek2 Department of Chemistry and Biochemistry, University of South Carolina [A, B., K. E. C.], and Department of Pathology, University of South Carolina School of Medicine [L. P.], Columbia, South Carolina 29208, and the Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892 [0. 0.] Abstrad Similar cellular responses are elicited by retinoic acid (RA) and transforming growth fador (TGF-fl). We investigated the ability of RA to modulate the produdion of TGF-fl in normal human keratinocytes (HKc) and HKc lines immortalized by transfedion with human papillomavirus type 16 DNA (HKc/HPV1#{212}). RA treatment of both normal HKc and HKc/HPV16 resulted in a 2-3-fold indudion in secreted levels of latent TGF-$. The indudion in TGF- secretion by LA was dose dependent, with significant increases observed with LA concentrations as low as 1-10 ntvi, and time dependent, with maximal indudion occurring about 3 days after initiation of RA exposure. In addition, RA induced intracellular levels of TGF-$ almost 5-fold. Sandwich enzyme-linked immunosorbent assays were used to specifically quantify TGF-$1 and TGF- 92 secreted by normal HKc and HKc/HPV16 cultured in the absence or presence of RA. RA increased the secreted levels of latent TGF-$1 and TGF- fl2 an average of 2- and 5-fold, respedively, with no major differences in the fold indudion between normal HKc and HKc/HPV16. Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced specific transcripts for TGF-$1 and TGF-i92 about 3- and 50-fold, respedively. RA treatment of HKc had no significant effed on the binding affinity of TGF- for its receptors or receptor number. Normal HKc and HKc/HPV16 displayed similar dose-dependent inhibition of proliferation by TGF-fl1. These studies indicate that RA may regulate growth control in both normal HKc and HKc/HPV16 by enhancing TGF- 1 and TGF- 2 produdion, which, after adivation at the cell surface, could inhibit cellular proliferation in an autocrine and/or paracrine manner. Introdudion Vitamin A or compounds which possess vitamin A-like activity, the retinoids, are essential for the proper growth and differentiation of epithelia in general and regulate the normal program of squamous differentiation in the skin (1 -3). Exposure of cultured keratinocytes to RA,3 the most potent of the known naturally occurring analogues of vitamin A, results in an alteration of a variety of cellular responses related to growth and differentiation. For ex- ample, retinoid treatment of keratmnocytes inhibits the expression of biochemical markers of terminal squamous differentiation (1-3) and inhibits the growth of rapidly proliferating cultures of keratinocytes (4-7). RA also mod- ulates the expression of enzymes and proteins involved in the formation and remodeling of the extracellular matrix. For example, RA inhibits the secretion of colla- genase by human keratmnocytes (8) and inhibits the expression of the metalloproteinase stromelysmn in poly- omavirus-transformed rat embryo fibroblasts (PyT2l) (9). On the other hand, the production of extracellular matrix components such as collagen type IV (a,) and laminin Bl are increased during RA-induced differentiation of F9 mouse teratocarcinoma stem cells (10). The mechanism by which retinoids control growth, differentiation, and extracellular matrix synthesis and turnover appears to be mediated through specific nuclear RARs that belong to the steroid and thyroid hormone receptor superfamily (1 1 -1 3). The RARs, like the steroid hormone receptors, are ligand-activated transcription factors. It is likely that the RARs are transcriptional me- diators of at least some of the biological responses elic- ited by RA in keratinocytes. Three different RARs have been identified, RAR-a, RAR-/ , and RAR-’y (1 1 -1 7). RAR- ‘V is the predominant RAR expressed in the epidermis, suggesting that it mediates the majority of RA responses in the skin (17, 18).