Structural Biochemistry/Cell Signaling Pathways/Transforming Growth Factor Beta

Overview
Transforming growth factor beta (TGFβ) is a small cell-signaling protein molecule (cytokine), secreted by the glial cells of the nervous system as well as some cells of the immune system. They are responsible for cell proliferation (cell growth), cell differentiation, apoptosis (cell death), and cell migration.

There exists three different isoforms of TGFβ, TGFβ-1, TGFβ-2 and TGFβ-3. The three forms of TGFβ are expressed by many different cells of the body and likewise most cells in the body also possess the TGFβ receptor sites for binding.

TGFβ is always produced in the form of an inactivated complex. In order for TGFβ to bind to its receptor and act on its function, it must be activated. Integrins which acts like a mediator between cells and tissues surrounding it are actually responsible for the activation of the inactive TGFβ complex to an active complex. The reason TGFβ is produced in its inactive form is so that its effects only occur at the right time and place. The function of active TGFβ is to bind to its receptor, which leads to a signaling cascade, and results in the activation or repression of gene transcription [1]. These pathways and the biochemical mechanisms that regulate them are of interest because of their importance to human health and role in diseases. Further understanding of the integrins that control TGFβ activation could lead to new therapeutics. Examples of such beneficial therapeutics include treating cancer because TGFβ has abnormal function during tumor formation [1].

Each of the three TGFβ isoforms contains a 25kDa N-terminal propeptide, which is called latency-associated peptide (LAP) as well as a 12.5 kDA C-terminal active TGFβ. The N-terminal LAP and C-terminal TGFβ are encoded by separate genes and together, form a homodimeric LAP- TGFβ propeptide complex. A propeptide is a short sequence that dictates how its associated protein folds [3]. In order for TGFβ to function, the LAP and TGFβ must be cleaved in the Golgi. However, the TGFβ receptors are blocked by cleaved LAP- TGFβ, leaving TGFβ in its inactive form [1]. The LAP- TGFβ complex is called the small latent complex (SLC). The covalent association of latent TGFβ and SLC forms the large latent complex (LLC). This has important effects when TGFβ localizes in the extracellular matrix and is activated by cells [1]. Studies have shown that some hydrophobic regions in LAP play an important role in the formation of latent TGFβ [4]. Because of its control over TGFβ, it is crucial that LAP does not contain mutations, because these can lead to disease when TGFβ function is not properly controlled [1].

Many different processes have been advanced to account for the activation of TGFβ, these include heat, acidic pH, reactive oxygen species, various proteases, the membrane glycoprotein thrombospondin-1 and shear stress, but however strong compelling arguments have come to show that integrins are responsible for the activation of TGFβ. Integrins are part of a cell adhesion and signaling receptor family. They are composed of an α and a β subunit that together create a heterodimeric transmembrane receptor I. There are a total of 24 integrin receptors in mammals formed by 18α and 8 β subunits. [5].                 Once activated TGFβ binds to its transmembrane receptors, TGFβRI and TGFβRII. These two receptors form complexes with TGFβ to enable its signaling. A simple pathway of TGFβ signaling can be shown on the figure below. Figure 1 shows that when TGFβ binds to TGFβRII, which subsequently binds with TGFβRI, intracellular signals are triggered and lead to activation or repression of transcription. TGFβ must be in its active form to bind with TGFβ RII. When TGFβ binds to TGFβ RII, TGFβRII phosphorylates TGFβRI. Then, Smad2 or Smad3 is phosphorylated by TGFβRI. Phosphorylated Smad2 or Smad3 can associate with Smad4. The resulting complex can translocate to the nucleus to activate transcription or induce apoptosis [1].

Figure 1. TGFβ Signaling Pathway

Activation of TGFβ
Activation of TGFβ by different intergrins can change the function of TGFβ. In mammals, six out of the 24 integrin can bind to latent TGFβ through a tripeptide RGD integrin-binding motif in the Latency-associated peptide (LAP) region in the latent complex. Integrins αvβ3 and αvβ5, when expressed in certain fibroblastic cells, activate TGFβ to cause lung fibroblast cells to differentiate into myofibroblast cells. αvβ6-mediated TGFβ activation helps to maintain the immune homeostasis in epithelial cells. Activation of TGFβ by αvβ6 has been proposed to play a role in cancer progression through tumor invasion via up-regulation of MMP9, but further studies are required to understand the process. TGFβ activated by αvβ8 regulates the brain vascular developement and, and when αvβ8 activates the isoform TGFβ1 and TGFβ3, the cytokin helps to control neurovascular development. Integrin αvβ8 activated TGFβ in dendrite cells also play a role in controlling self-harful T-cell responses. The function of TGFβ activated by integrins αvβ1 and α8β1 have yet to be determined, and requires further study.

Integrins can bind to TGFβ in two different ways. Integrins (like αvβ5 and αvβ6) bind to the LAP region outside the cell, and from the generation of a pulling force by the actin cytoskeleton connected to cytoplasmic domain of the integrin, there is a conformational change. This conformational change of the TGFβ complex also requires the binding of the LTBP1 to ECM, which creates a holding force for the conformational change. The mechanism of the conformational change of the latent complex can be improved by pathways that enhance cell contraction, such as thrombin. This will lead to the activation of TGFβ. There is little knowledge past this extent of the mechanism of the conformational change since there is no structural information currently available for an integrin-latent TGFβ complex.

The second way an integrin can bind to the TGFβ is through cell-specific mechanisms. For example, in lung and airway cells, αvβ8 binds to TGFβ when the cell surface metallprotease MT1-MMP proteolytically cleaves the LAP region. This protease-dependent mechanism has not been tested in other αvβ8-expressing cell types that are known to activate TGFβ, so it is unsure if this is the mechanism αvβ8 induced TGFβ activation.