Ralf Weiskirchen, Steffen K. Meurer, Olav A. Gressner, Jens Herrmann, Erawan Borkham-Kamphorst, Axel M. Gressner

Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Germany

FIGURES

Figure 1. The TGF-beta superfamily. Based on their structural features the 35 mammalian members of the TGF-beta family are subdivided into (i) TGF-betas, (ii) activins/inhibins, (iii) bone morphogenetic proteins (BMPs)/growth and differentiation factors (GDFs), and (iv) the more distantly related group of GDNF ligands. The affiliation to one of these subgroups is ambiguous and handled somewhat irregular. TGF-beta₁, BMP-7, BMP-2 and BMP-4 (all marked in red) are those cytokines with outstanding importance in control of fibrogenesis.

Figure 2. Structural features of BMP-7 and TGF-beta₁. (A) Both, BMP-7 and TGF-beta₁ share the same modular structure typical for members of the TGF-beta superfamily. The N-terminal leader sequence (in red) is necessary for targeting to endoplasmic reticulum (ER) and subsequent cellular secretion. The latency-associated peptide (in blue) is required for masking (latency), stabilization and extracellular matrix deposition. The mature (biologically active) peptide (in green) is located at the C-terminus of the pre-propeptide. Locations of amino acid positions are given for human BMP-7 (Swiss-Prot P18075) and TGF-beta₁ (Swiss-Prot P01137). (B) Sequence alignment of human TGF-beta₁ and BMP-7. The regions of the signal region, latency-associated peptide, and mature cytokine are given in red, blue, and green, respectively. (C) Schematic overview about sequence similarities between BMP-7 and TGF-beta₁. The highest degree of similarity (~40-70%) of both cytokines is found at the C-terminal regions harboring the mature peptides.

Figure 3. Two- and three-dimensional structure of mature TGF-betas and BMPs. (A) Members of the TGF-beta/BMP/GDF family are only active as dimers in which the individual monomers are linked by a single disulphide bond that is in human TGF-beta₁ located at position 77 of the mature peptide (position 356 from the Start-ATG). In addition, the TGF-beta monomers contain four intrachain disulphide bonds (solid lines) that are conserved in TGF-betas and inhibin-betas, while the other members of this family lack the first bond (speckled line). The dimers are bridged by one intermolecular disulphide bond (dotted line). The sequence positions of cysteines involved in disulphide bonding correlate to those counted from the beginning of human mature TGF-beta₁ (for orientation refer to Figure 2B). (B, C) The tight network of intramolecular disulphide bonds and the single intermolecular linkage of two monomers cause the butterfly-like tertiary fold that is typical for dimers of the TGF-beta family. For this analysis the minimized average nuclear magnetic resonance (NMR) structure of human TGF-beta₁ (B) or the crystal structure of human BMP-3 (C) that are deposited in the Brookhaven Protein Databank (PDB) under accession numbers 1KLC and 2QCQ, respectively, were taken for molecular visualization using the RasMol program (Windows version 2.7.4.2). For more structural details, refer to the original literature describing the respective three-dimensional structures of these TGF-beta superfamily members (151, 152).

Figure 4. TGF-beta/BMP receptors. (A) The type I receptors are further grouped into three subgroups. The ALK-1 group contains ALK-1 and ALK-2, the ALK-6 group contains ALK-3 and ALK-6, while the ALK-5 group contains ALK-4, ALK-5 and ALK-7. The protein sequences of the different ALKs from rat were taken as input for this dendrogram. (B) The type II receptors BMPRII, ActRIIA and ActRIIB are specific for BMPs, while TGFbetaRII is specific for TGF-betas. In this dendrogram the following proteins were aligned: human ActRIIA (hActRIIA), human ActRIIB (hActRIIB), human TGFbetaRII, murine BMPRII (mBMPRII), human Mast cell immunoreceptor signal transducer (hMIST-II), Drosphophila melanogaster receptors Wishful Thinking (dwit) and Punt (dpunt), and Caenorhabditis elegans Cell surface receptor of the abnormal dauer formation family member (cdaf-4).

Figure 5. Receptor for BMP-7 and TGF-beta. BMP-7 bind to the type II receptors BMPRII, ActRIIA and ActRIIB that subsequently activate with different specificities the type I receptors ALK-3, ALK-6 or ALK-2. In contrast, TGF-beta binds to TGFbetaRII that subsequently transphosphorylate ALK-1 and ALK-5.

Figure 6. Modulation of BMP/TGF-beta signaling by Endoglin. The accessory type III receptor Endoglin has affinity for individual members of the TGF-beta superfamily that influences the affinity for individual type II receptors. In the presence of Endoglin, BMP-7 signaling is increased.

Figure 7. BMP signal transduction. Signaling by members of the BMP-subfamily of ligands is initiated by binding to a heteromeric complex of type I receptors, e.g. ALK-2, ALK-3 and ALK-6, as well as type II receptors, e.g. BMPRII, ActRII and ActRIIB. The access of ligands to the receptors is regulated by secreted proteins like CTGF (negatively) or KCP (positively). In addition, type III receptors like Betaglycan or Endoglin modulate the signal transmission. Intracellular mediators belong to the family of MAP kinases (p38, Erk1/2; left) or to the Smad family (right), depending on the composition of the formed receptor complex. BRSmads are activated by phosphorylation through the corresponding type I receptor, associate with the co-Smad4 and translocate into the nucleus to regulate transcription of target genes in conjunction with co-repressors and co-activators. The signaling circuit (green) is controlled by the inhibitory Smad (i.e. Smad6) at several steps. The activated forms of type I receptors and Smads are deactivated through dephosphorylation by specific phosphatases and these components are marked for degradation with ubiquitin by the ubiquitin ligase Smurf. Abbreviations used are: CTGF: connective tissue growth factor; KCP: Kielin/Chordin-like protein; BMP: bone morphogenetic protein; ECD: extracellular domain; TM: transmembranal domain; KD: kinase domain; P: phosphate; Ubq: ubiquitin; MH1/MH2: MAD homology domain 1/2; TF: transcription factor

Figure 8. Complex pattern of interaction between TGF-b and BMP-7. BMP-7 supports the epithelial phenotype by inducing the expression of Smad7 through the Smad1/4/GATA complex that inhibits TGF-b signaling, and Id2/3 which inactivate the repressor E2A to permit expression of E-cadherin. Ids also inhibit several TGF-b responsive genes including CTGF and alpha -SMA, Col 1 and PAI-1. On the other hand, TGF-beta supports the mesenchymal phenotype by the rapid induction of CTGF. CTGF binds BMP-7 and inhibits BMP-7 signaling as evidenced by lower levels of pSmad1/5 and Id1 mRNA (140). In addition, CTGF activates several receptor systems that integrate with TGF-b /Smad signaling leading to the induction of transcription repressors that inhibit E-cadherin. CTGF further inhibits the expression level of Smad7, thus enhancing the transcription of TGF-beta-responsive genes. Moreover CREB, activated via CTGF, associates itself with the BMP/Smad complex, activating expression of Smad6 that not only inhibits the BMP-7 signaling pathway but also Id2/3 activity that leads to repression of E-cadherin. TGF-beta-induced PAI-1 in turn inhibits the activities of uPA and tPA that can activate the MMPs. Integrin-linked kinase (ILK), an intracellular serine/threonine kinase, associates with beta-integrin and regulates E-cadherin at the transcriptional level via the transcriptional repressor SNAIL-1. In addition, ILK phosphorylates Akt (PI3K) and glycogen synthase kinase (GSK), phosphorylation of GSK-3 resulting in nuclear translocation of beta-catenin and activation of the Wnt signaling pathway, which has also been strongly implicated in EMT.