Role of TGF-β3 in the regulation of immune responses T. Okamura1,2, K. Morita1, Y. Iwasaki1, M. Inoue1, T. Komai1, K. Fujio1, K. Yamamoto1 1Department of Allergy and ABSTRACT ences, indicating their non-redundancy Rheumatology, Graduate School of Transforming growth factor-betas (12-16). The TGF-βs have opposite ef- Medicine, The University of Tokyo, (TGF-βs) are multifunctional cytokines fects on tissue fibrosis. Wound-healing Tokyo, Japan; that have been implicated in the regu- experiments revealed that TGF-β1 2Max Planck-The University of Tokyo Center for Integrative Inflammology, lation of a broad range of biological and TGF-β2 cause fibrotic scarring The University of Tokyo, Tokyo, Japan. processes, including cell proliferation, responses and that TGF-β3 induces a Tomohisa Okamura, MD, PhD cell survival, and cell differentiation. scar-free response (17). Both TGF-β1 Kaoru Morita, MD The three isoforms identified in mam- and -β2 activate the collagen α2 (I) Yukiko Iwasaki, MD, PhD mals, TGF-β1, TGF-β2, and TGF-β3, gene promoter, resulting in increased Mariko Inoue, MD have high sequence homology, bind to collagen synthesis (18). It was also re- Toshihiko Komai, MD the same receptors, and show similar ported that TGF-β1, but not TGF-β3, is Keishi Fujio, MD, PhD biological functions in many in vitro a crucial factor in the development of Kazuhiko Yamamoto, MD, PhD studies. However, analysis of the in vivo pulmonary fibrosis (19). Most of the in- Please address correspondence to: functions of the three isoforms and mice formation on the immunological activ- Keishi Fujio, MD, PhD, deficient for each cytokine reveals strik- Department of Allergy and ity of TGF-βs derives from studies of Rheumatology, ing differences, illustrating their unique TGF-β1 and, in part, TGF-β2, whereas Graduate School of Medicine, biological importance and functional recent investigations have begun to il- The University of Tokyo, non-redundancy. Although increasing luminate the importance of TGF-β3 in 7-3-1 Hongo, Bunkyo-ku, evidence suggests that TGF-β1 and, to the immune system. In this review, we Tokyo 113-8655, Japan. a lesser extent, TGF-β2 play an integral focus on recent novel insights into the E-mail: [email protected] role in maintaining immune tolerance, immunological role of TGF-β3, with Received and accepted on August 28, 2015. the immunological role of TGF-β3 has comparison to TGF-β1 and -β2. Clin Exp Rheumatol 2015; 33 (Suppl. 92): not been carefully evaluated. Recent S63-S69. studies have focused on the multifunc- TGF-β family synthesis © Copyright CLINICAL AND tional role of TGF-β3. In this review, The three isoforms of TGF-β each uti- EXPERIMENTAL RHEUMATOLOGY 2015. we provide an overview of the role of lise a unique activation mechanism TGF-β3 in immunity, with comparison from their latent forms (20). TGF-βs are Key words: TGF-β3, TGF-β1, to TGF-β1 and -β2. initially synthesised as larger precursor TGF-β2, humoral immunity, polypeptides containing a hydropho- autoimmune disease, CD4+CD25- + Introduction bic signal sequence, latency-associated LAG3 regulatory T cells, Egr-2 Three structurally similar isoforms of peptide (LAP), and mature peptide (pre- TGF-β (TGF-β1, TGF-β2, and TGF-β3) pro-TGF-β). Pre-pro-TGF-β monomers have been identified in humans (1). dimerise through disulfide bridges be- TGF-β1 was first cloned from a human tween cysteine residues in LAP and cDNA library (2) and human TGF-β2 cysteine residues in the mature peptide and TGF-β3 cDNA were subsequently (pro-TGF-β), and are cleaved by furin cloned (3, 4). TGF-β1 is highly homol- convertase but remain non-covalently ogous to the TGF-β2 and -β3 isoforms associated with the mature peptide. (5). TGF-β2 and -β3 share 71% (β2 vs. LAP confers latency to the mature pep- Competing interests: β1) and 76% (β3 vs. β1) sequence iden- tide by shielding the receptor-binding K. Yamamoto has received financial support or fees from AbbVie, Astellas, tity to TGF-β1 (6). Although TGF-β4 region of active TGF-β (21). Similar BMS, Daiichi-Sankyo, MitsubishiTanabe, and -β5 were identified from cDNA li- latent complexes have not yet been re- Pfizer, Sanofi, Santen, Takeda, Teijin, braries of chicken chondrocyte and frog ported for other members of the TGF-β Boehringer Ingelheim, Chugai, Eisai, oocyte, respectively (6, 7), mammalian superfamily. Ono, Taisho Toyama, UCB, Immuno- TGF-β4 and -β5 have not yet been de- The activity of secreted TGF-β is pri- Future, Asahi Kasei and Janssen; scribed. In vitro experiments indicated marily regulated by the conversion K. Fujio has received financial support or that TGF-β1, -β2, and -β3 often exhibit of its latent form to its active form. fees from Astellas, BMS, Daiichi-Sankyo, MitsubishiTanabe, Pfizer, Santen, Takeda, similar biological activities (8-11). Although tissues contain significant Chugai, Eisai, Taisho Toyama, UCB, and Despite this high sequence homology, quantities of latent TGF-β, the activa- Janssen; the other co-authors have analysis of the in vivo functions of the tion of only a small fraction of this la- declared no competing interests. three isoforms reveals marked differ- tent TGF-β exerts maximal effects on S-63 Role of TGF-β3 in the regulation of immune responses / T. Okamura et al. target cells. In lymphoid tissues, LAP whereas αvβ8 integrin is expressed on endothelial cells (42). TGF-β3 induces is thought to be removed by throm- most leukocytes. Leucocyte-specific the activation of TGF-β receptor II ki- bospondin, plasmin, and acidification conditional deletion of Itgb8 (which nase and phosphorylates ALK5, which during the inflammatory process (22). encodes integrin β8) causes severe recruits ALK1 into a TGF-β receptor The TGF-β-LAP complex, called the inflammatory bowel disease and the complex. ALK5 kinase activity is re- small latent complex (SLC), is further development of high levels of auto- quired for optimal ALK1 activation. bound to latent TGF-β-binding protein antibodies against double-stranded Activated ALK1 and ALK5 induce (LTBP). There are four isoforms of DNA (32). These findings indicate the the phosphorylation of Smad1/5 and LTBP in humans (LTBP-1, -2, -3, and importance of αvβ8 integrin-mediated Smad2/3, respectively, whereas ALK1 -4). LTBP-2 is unique in the LTBP fam- TGF-β activation by lymphoid cells directly inhibits ALK5/Smad3 signal- ily as it is the only isoform that does for preventing the immune dysfunction ling (41). Intriguingly, endoglin, a co- not bind to latent TGFβ (23). TGF-β- that results in inflammatory bowel dis- receptor for TGF-β1 and TGF-β3, func- LAP-LTBP is termed the large latent ease and autoimmunity. The expression tions as a modulator of the balance be- complex (LLC). The LTBP is not nec- of αvβ8 integrin on dendritic cells and tween TGF-β/ALK1 and TGF-β/ALK5 essary for latency (24), but it does play activated CD4+CD25+Foxp3+ regulato- signalling. Endoglin, which stimulates a critical role in the assembly and secre- ry T cells (Tregs) (32, 33) might play a TGF-β/ALK1 signalling and indirectly tion of TGF-β (25), as well as target- key role in promoting immune regula- inhibits TGF-β/ALK5 signalling, regu- ing the LLC for storage through inter- tion by activating TGF-β1. Consistent lates endothelial cell proliferation (43). actions with fibronectin and fibrillin in with these reports, Itgb8-deficient mice These findings suggest that the ratio be- the extracellular matrix (ECM) through show perinatal lethality with profound tween ALK1 and ALK5 signalling may non-covalent interactions (26). LTBPs defects in vascular development, par- provide one explanation for the bifunc- covalently bind to the LAP portion of ticularly in the brain (34), and a simi- tionality of the TGF-βs. Smad-inde- SLC through the third eight-cysteine lar phenotype is also seen in mice with pendent non-canonical pathways medi- type domain of LTBP. The third eight- mutation in Tgfb1 or deletion in the ated by TGF-β have also been reported. cysteine type domain of LTBP-1 and Tgfb3 gene (35). TGF-β1 induces profibrotic signalling LTBP-3 can associate efficiently with by activating tyrosine-protein kinase pro-TGF-β1, -β2, and -β3. Conversely, TGF-β family receptors and signal c-Abl, which is a mediator for fibrotic LTBP-4 binds to TGF-β1-LAP more transduction responses (44). Several studies have weakly than the other two LTBPs, indi- TGF-βs are secreted as latent proteins. suggested other possible non-canonical cating that LTBP-1 and LTBP-3 might After activation, TGF-βs interact with pathways, such as mitogen-activated be the primary proteins responsible for the same receptor heterodimers, TGF-β protein kinases (MAPK), phosphati- binding to SLC (27). The C-termini of receptor I (TGFBR1/ALK-5) and dylinositol-4,5-biphosphate 3-kinase LTBP-1, -2, and -4 bind to a similar TGF-β receptor II (TGFBR2), to acti- (PI3K), AKT, and the NF-κB pathway region in the N-terminus of fibrillin-1 vate the canonical Smad2/3 signalling (45). More extensive studies of TGF-β and -2 through non-covalent interac- pathway. However, TGF-β2 requires signal transduction in immune cells are tions, whereas LTBP-3 reportedly does binding of TGF-β receptor III (TGF- needed to elucidate the specific role of not (28). However, LTBP-3 co-localises BR3) prior to biding TGFBR2 (36). each TGF-β isoform in the immune with fibrillin fibres in tissues, and loss TGF-β3 transmits signals biologically system. of fibrillin-1 prevents the incorporation similar to and more potently than those of LTBP-3 into a matrix, indicating that transmitted by TGF-β1 and -β2, de- TGF-β-deficient animals LTBP-3 association with the matrix de- pending on the cell type and assay used TGF-β1-deficient mice die by 4 weeks pends on fibrillin-1 microfibrils (29).