Organ
of the
GD — Society for Dermopharmacy
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Issue 1 (2002) |
Dermatotherapy
Transforming Growth Factor β (TGF-β)
The transforming growth factor (TGF-β) belongs to a family of 40 structurally similar polypeptide growth factors regulating a fascinating diversity of cellular processes as apoptosis, proliferation as well as differentiation, adhesion and agility. The clearing up of the signal cascades of TGF-β as well as the knowledge of their physiological significance open up perspectives for the development of new specifical drugs. For TGF-β respectively TGF-agonists, the application at hyper proliferating skin diseases as psoriasis vulgaris seems to be useful whereas TGF-antagonists as means for the enhancing of the wound healing can be taken into consideration.
With psoriasis vulgaris, the most frequent hyper proliferating skin disease, besides an enhanced division rate and a disturbed differentiation of the keratinocytes also a leukocyte infiltration of the epidermis is entailed. Due to the fact that TGF-β inhibits the growth of epidermal cells and furthers their differentiation, a disturbance in the signal cascades of TGF-β seems to be involved in psoriatic processes. However, the intracellular signal paths of the substance in keratinocytes have so far only been subject to minor investigations. This is why the findings known so far are presented in the following.
The TGF-β family comprises besides the TGF-β also the activine and BMP (Bone Morphogenetic Protein)-sub-family. These sub-families are often closely related and are present in nearly all zoological species. The prototype TGF-β has got its denomination because it was discovered as factor that generated a transforming phenotype in the fibroblasts of rodents.
Signal Paths of TGF-β
TGF-β is of particular interest from a dermatological point of view. It has been proven that it does not only have an antiproliferating and differentiating effect on epidermal and haematopoietic cells but also furthers the wound healing as well as the embryo- and angiogenesis. Moreover, TGF-β increases the immunological properties of neutrophile granulocytes and monocytes.
All effects are performed via membrane-bound glycoproteinreceptors (type 1 and 2), which both have a serine/threonine-kinase-activity and are specific for the different members of the TGF-β -family. Subsequent processes are only released if both receptors form a complex by means of a bridging with TGF-β . In the course of this process, the TGF-β links to the type-2-receptor first and enables thus the approach of the type-1-receptor. Only after the combining of the receptors, the kinase activity of the type-1-receptor is "switched on", and so-called Smad-proteins are able to accumulate in order to be phosphorylized by it. They in fact transmit the TGF-β -transferred signals to the nucleus, the "TGF-β genes", in the end, their actual target, (figure 2, page 16).
| In the column "Dermatotherapeutics on the Horizon" we present in irregular intervals the pharmacological profile of substances, which could be introduced in Dermatotherapy one day. Pharmacist Bettina Sauer, employed as doctorand of processor Dr. Monika Schäfer-Korting at the department pharmacy at the Free University of Berlin, is the responsible editor of this series. After the presentation of the macrolid Sirolimus and the monoclonal antibody Infliximab in DermoTopics no. 1 respectively no. 3 (2001), this issue deals with the perspectives of the polypeptide growth factor TGF-β respectively of its agonists and antagonists. TGF-agonists represent a possibility for the treatment of hyper proliferating skin diseases as psoriasis vulgaris whereas TGF-antagonists could gain increasing importance as means for the enhancement of wound healing. |
Smad-Proteins and their Signal Paths
The name of Smad-proteins is derived from the genes encoding them. They had been identified in genetical studies at drosophila and C. elegans for the first time. The drosophila-gene is designated as Mad (Mother against decapentaplegic), the gene in C. elegans as Sma (Small body size). The combination of these two designations creates the name "Smad". Structural and functional three sub-families of the Smad-proteins are distinguished (figure 1), for all three of which a similarly strong preserved basic sequence is characteristic.
The receptor-Smad proteins (R-Smads, Smad 2 und 3) directly interact with the type-1-receptor activated by the mechanisms as described above. Only after the phosphorylization the R-Smads are able to bind the cytoplasmatic cooperative Smad-proteins (Co-Smads, Smad 4), which in the end serve for the accumulation of the Smad-complexes at the DNA-promoters and the transcription activation.
The third group are the inhibitory Smad-proteins (I-Smads, Smad 6 and 7). They are characterized by striking structure variations in comparison with the R- and Co-Smads. Thereupon can be attributed that they competitively antagonize the TGF-transferred signal transduction. Both I-Smads are increasingly developed due to a large offer of growth factors, serve therefore as negative feedback. They are able to compete with the R-Smads both for receptor binding and prevent the interaction of R-Smad and Co-Smads.
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| Figure 1: Smad-Proteins and interactions between the representatives of the three sub-families (please refer to the text for explanations) |
Structure of Smad-Proteins
All Smad-proteins have a relatively similar structure. The high-preserved chain ends are connected by means of a linker of variable length and sequence. The N-terminus of Smad 4, the so-called MH1-domain, takes on the function of attachment to DNA-promoters after activation and translocation into the nucleus. The C-terminal MH2-domain is able to link to various proteins, for example to the type-1-receptors, to other Smads and to transcriptions factors.
The Smad-complex permeates after phosphorylization into the nucleus, where it enters into interactions with transcription factors via the MH2-domain of the R-Smads or directly arranges by attachment of the MH1-domain of Smad 4 to promoters the gene expression.
As example for the latter, the genes p15 and p21 are mentioned. Both encode cycline-related kinase inhibitors, which stop the cell cycle and thus decelerate the proliferation. There are also linking positions at the promoters of the I-Smad genes for Smads serving the high-regulation of the I-Smads. Smad-complexes seem, however, to also have an inhibitory effect on DNA-segments of growth-stimulating genes as c-myc.
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| Figure 2: The cellular signal path of TGF-β is very complex: TGF-β links to the membrane-bound type-2-receptor and allows thus the adsorption of the type-1-receptor. Then TGF-β activates the kinase function of the type-2-receptor with subsequent stimulation of the kinase-function of the type-1-receptor. The so-called receptor-Smad-proteins (Smad 2 and Smad 3) link to the receptor complex and are phosphorylized by the type-1-receptor. The Smad- anchor protein SARA enhances the adsorption of the R-Smads. The phosphorylized R-smad-proteins form a complex with the cooperative Smad 4. It is able to penetrate the nucleus. Here the activated R-Smads adsorb at DNA-promoters and/or transcription factors and control transcription processes. The inhibitory Smad-proteins (Smad 6 and Smad 7) antagonize the adsorption of the R-Smads at the receptor complex or at Smad 4 (please refer for further explanations to the text). |
For the consolidation of Smad-proteins with transcription factors, the Activine Responsive Factor (ARF) is indicated. This factor is only able to interact with the Activine Responsive Element (ARE) at the Mix2-Gene after the cooperation with the Smad-complex.
Defects of TGF-β and Smad-Signal Paths
In the course of the increasing establishing of the knockout procedure and its application also on Smad-proteins and TGF-β it became possible to observe the repercussion of the lack of these signal molecules on the regular skin development. For this intervention, which deliberately eliminates the genes for certain proteins, the blastocyte forming after the natural conception is removed and transfected with foreign-DNA. The insertion of the DNA is effected by transfer of retroviruses or microinjection. Following, the embryonic cells are implanted with a surrogate mother and their development is observed. The added DNA, which reaches by itself the nucleus from the cytoplasm, contains a segment, which exactly corresponds to the gene to be eliminated. This segment accumulates in the course of the homological recombination at the target sequence and fuses with it at the superimposed position. As during this process the entire carrier structure is incorporated into the gene, transcription and the protein synthesis resulting are no longer possible.
Various working groups have eliminated Smad 2, 3 and 4 as well as TGF-β . Only the Smad-3-knockout mouse survives the embryogenesis. In case of a lack of Smad 2 or Smad 4, the regular formation of germ layers does not take place. The Smad-5-knockout mouse dies on 10th embryonic day, as it is not able to develop a blood vessel system.
When eliminating Smad 3, an immunological dysregulation in form of a severe abscess and fistula development shows in the mucosa layers. The mice succumb at the age of three weeks at the latest. Before a substantial hyper proliferation of keratinocytes comes about which detach from skin as scales. TGF-β is no longer able to unfold its anti-proliferative effect at these tissues. These results are in accordance with the reduced expression of TGF-β -receptors at psoriasis and show the significance of TGF-β and Smad 3 in the treatment of hyper proliferating diseases.
Influence of TGF-β on the Wound Healing
TGF-β , being a wound-healing stimulating cytokine, it is astonishing that Smad-3-knockout mice show a significantly accelerated wound healing after having suffered injuries. The wound-healing process is characterized by a release of TGF-β from degranulating thrombocytes, which are migrating into the wound area. TGF-β produces a strong migration of monocytes and neutrophiles into this region. They eliminate microorganisms, keep the wound edge clean, and enhance, however, also local inflammations by the release of cytokines and proteases - an unfavorable process for the wound healing.
Also fibroblasts perform TGF-β -stimulated a chemo taxis, proliferate substantially, further the wound edge contraction for a better closure and secrete matrix material as collagen and fibronectine for the tissue reconstruction. TGF-β activates Smad-produced the transcription of the genes for collagen and fibronectine. The leukocytes and fibroblasts migrated to the wound area secrete TGF-β again. Thus its tissue level rises increasingly and enhances the migration until a compensatory down-regulation of Smad 3 occurs. Only then the activity of the described cells decreases. Simultaneously TGF-β level and proliferation inhibition of the keratinocytes are cancelled. Finally a re-epithelization of the wound takes occurs.
The accelerated wound healing in case of a lack of TGF-β respectively down-regulation of Smad 3 is on one hand probably caused by the enhanced keratinocyte proliferation. On the other hand the reduced monocyte infiltration, which does not exercise a negative effect at lacking wound contamination, prevents the development of inflammations. These two factors seem to overcompensate the reduced matrix formation. The wounds of Smad-3-knockouts heal within two days, whereas wild type-mice require on average four to five days for this process. However, the assessment of this fact remains difficult. If the matrix formation is underrepresented at a Smad-3-shortage, this may adversely affect the solid new tissue. Furthermore, the defence mechanisms with bacterial contaminations are insufficient without the migration of immunocytes.
Perspectives for new Medicinal Substances
The clearing up of the signal cascades of TGF-β as well as the knowledge of its physiological significance seem to be of importance for the development of new specifical medicinal substances. TGF-agonists could be applicable with hyper-proliferating skin diseases as psoriasis, TGF-antagonists with wound healing. Although TGF-β is so far not available in any form of presentation, its use as drug is absolutely to be taken into consideration. The same applies to active substances modifying the TGF-β metabolism or having an impact on the TGF-β -signal path. In this context, substances are a possibility, which stimulate or inhibit receptors or also those modulating Smad-proteins. The multitude of intervention possibilities on the various steps of the signal cascade of TGF-β opens up promising perspectives in search of new active substances.
Literature
[1] Ashcroft GS, Yang X, Glick AB, Weinstein M, Letterio JL, Mizel DE, Anzano M, Greenwell-Wild T, Wahl SM, Deng C, Roberts AB: Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat. Cell Biol. 1 (1999) 260-266
[2] Itoh S, Itoh F, Goumans MJ, Ten Dijke P: Signaling of transforming growth factor-beta family members through Smad proteins. Eur. J. Biochem. 267 (2000) 6954-6967
[3] Massague J: TGF-β signal transduction. Annu. Rev. Biochem. 67 (1998) 753-791
[4[ Yang X, Letterio JJ, Lechleider RJ, Chen L, Hayman R, Gu H, Roberts AB, Deng C: Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness toTGF-β. Embo. J. 18 (1999) 1280-1291
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