Landmark experiment of the discovery of Transforming Growth Factor beta (TGF β) dates to 1981. Virally transformed cell lines released Sarcoma Growth Factor(SGF) like protein with an approximate molecular weight of 10kda. SGF like proteins were equally potential to take part in transformation (hence named transforming growth factors -TGF) and growth pattern of a cell (1). SGFs were characterised by high-pressure liquid chromatography (HPLC) into two subsets, TGF α and TGF β. TGF α cannot transform the cells efficiently in the absence of TGF β; however, TGF β alone cannot bring out the transformation. Thus, the activity of TGF β is thought-provoking with its role in both tumour suppression and activation (2,3).
Canonical TGF β signalling, also called Smad signalling involves the Smad (Mothers against decapentaplegic homolog) proteins which transduce the signal to activate a wide range of transcription factors in the nucleus. Eight distinct Smad proteins are identified and are functionally classified into three types; Receptor-regulated Smad (R-Smad), Co-mediator Smad (CoSmad) and the Inhibitory Smad (ISmad). Smad 4 (Co-Smad) is the outcome complex from activated Smad and is forwarded to bring out activation of a transcription factor. Smad6/7 is Inhibitory and competes with R-Smad for the receptor and activates Smurf (Ubiquitin ligase) to degrade activated Smad 4. TGF β can initiate the signal transduction of other pathways independent of Smad called Non-Smad signalling. Different ligands and different receptor belong to the same family can activate the TGF β signalling; each combination of ligand and receptor initiates the cytoplasmic proteins in a different manner and hence results in varied cellular response. Interaction of ligand-receptor is controlled by a special class of proteins called ‘ligand traps’ (4).
Figure 1: involvement of different ligand traps in bringing the ligands to the vicinity of the receptor, Different ligands are associated with specific receptors which in turn activate transducing protein complexes and carries the signal to the nucleus. Smad4 complex is also involved in this signal transduction cascade (4). TGF β signalling is at most important for wound healing, regeneration of damaged tissues and even in scar formation! Signalling process is highly organised and helps in maintenance of homoeostasis. The perhaps controlled inflammatory response is also a direct action formulated by the TGF β signalling cascade (5). Figure 2: Generalised scheme of TGF β signalling cascade. Ligand binding to the receptor dimerizes the cytoplasmic domain of the receptor complex and undergoes phosphorylation. Phosphorylation makes them Serine-Threonine kinase to activate Smad complex by itself becomes dephosphorylated. A hint of Non-Smad signalling is also depicted in the diagram which can also bring a similar cellular response. Smad 7 and Smurf complexes are the negative regulators of TGF β and hence the signalling is set under control (6). SARA (Smad Anchor for Receptor Activation) is a key protein which gathers Smad2 to the cytosolic domain of the receptor; which enhances the possibility for accurate phosphorylation. Epithelial-mesenchymal transition (EMT) can potentially modify the SARA activity (7). In spite of having lifesaving physiological functions, TGF β can even be fatal. Thus, TGF β signalling is designated as a double edged sword as it promotes both pro-oncogenic activation as well as an apoptotic cascade.
Tumour promotion through TGF β signalling: NMA, an orthologue of BAMB protein, blocks the TGF β signalling at an early stage. Overexpression of TGF β signalling and cut down in NMA expression is a clear demarcation in melanoma cells. This is one of the striking examples to illustrate the tumour promotion activity through TGF β signalling. Overexpression of TβRǁ in pancreatic cancer, Hyperactivity of R-Smad in colon cancer justifies the tumour promoting ability of TGF β. Mutations in Smad 2 promote invasiveness rather than blocking the signalling pathway (8). TGF β functions as a growth inhibitor in normal cells; whereas the tumour cells are destined to get the resistance to growth inhibition activity. On the other hand, TGF β secretion would become either autocrine or paracrine mode which would possibly increase the rate of cell proliferation and may even lead to metastasis. Mutations in the protein complexes involved in TGF β deviates the pathway into a tumorigenic. Overexpression of c-myc suppresses TGF β signalling or gives resistance to growth inhibition responses. Along with which c-myc also represses genes of p15 and p21 and thereby results in uncontrolled cell proliferation. The cytokine response may not shut down; in contrast, they exhibit epithelial to mesenchymal transformation which is the initial event in cancer progression and metastasis (9). Crystal clear results were obtained to explain the supportive function of TGF β in metastasis. Breast cancer cells named MDA-MB-231 tend to metastasize to bones. TGF signalling was blocked in these cells and was injected into immunodeficient mice which developed fewer tumours and sailed very fewer osteoclasts (10). Prostate carcinoma cells in immunodeficient mice expressed anomalous angiogenesis by the virtue of overexpressed TGF β signalling. Site-specific administration of antibodies of TGF β neutralised the signalling pathway and reduced the angiogenesis drastically. TGF β presumably activates angiogenesis inducing factor, Vascular Endothelial cell Growth Factor (VEGF) (11). Tumour suppression through TGF β signalling Initial characterization of TGF β signalling revealed the growth inhibition activity as a key cellular response. The cellular architecture was chiefly governed by TGF β signalling and hence could able to maintain the homoeostasis. In normal cells, TGF signalling was found to be very prominent and maintains a threshold level of TGF β. T-->C transition in 29th nucleotide of TGF β enhances the signalling efficiency by increasing its level in serum showed significantly reduced the risk of breast cancer by 50%. Investigation on keratinocyte cell line confirmed the role of TGF β in the maintenance of genomic stability. N-phosphonoacetyl-L-Aspartate treatment to the keratinocyte cells with and without TGF β was assessed and found that the cells with TGF β signalling lesser gene amplification. Restoration of autocrine activity of TGF β lead to drastic reduction in the activity of telomerase. Human colon carcinoma HCT116 cells were characterised after restoration and found that RNA levels of telomerase reverse transcriptase were greatly reduced. Scale down in the activity of telomerase spontaneously drags the cell to senescence. Thus, TGF β plays an indirect physiological role in suppression of cell growth (6). Figure 3: p15 is an effective inhibitor of cyclin-dependent kinases and results in cell cycle arrest. In the absence of TGF β signalling Myc and Miz-1 forms a complex which blocks the transcriptional activation of p15 gene and may lead to tumorigenesis. However, in presence of TGF β signalling, Smad complex disrupts Myc-Miz association and promotes the transcriptome of p15 (12). Smad 6 and Smad 7 are the major Inhibitory Smad which brings negative regulation of TGF β signalling. I-Smad has conserved C-terminal MH2 domain but lacks N-terminal MH1 domain; which are significantly used for phosphorylation in Co-Smad and R-Smad. Single molecule force spectroscopy revealed that even Smad 7 can bind to DNA at Smad Binding Element (SBE) CAGA box. The free N-terminal region of the Smad 7 binds to DNA but not MH2 domain; other Smad complexes bind to DNA with MH2 domain! Hyperactive Smad drastically reduces TGF β signalling and thus results in growth inhibition. On the other hand, Smad 7 activates Smurf1 and 2 to degrade either Smad complexes or ALK5/TβR1 receptor through ubiquitin ligase or proteasomal degradation pathway. Smad 6 functions in different mechanism and it acts as a transcriptional repressor by targeting Hoxc-8 or by binding to DNA or by effective activation of transcriptional corepressor, Histone deacetylase (13). Stronger tumour suppression could be done by forcing the cells to apoptosis; for which TGF β signalling is a highly targeted weapon. Programmed suppression of inhibitors of the intrinsic apoptotic pathway, BCL-X(L), triggers the activation of BIK complex through TGF β signalling. Smad complexes binds to consensus sequence to activate the transcription of BIK; meanwhile it inhibits BLC and sensitises Burkitt’s
Lymphoma (BL) cells to induce apoptosis through TGF β signalling. Thereby it evidently explains the involvement of TGF β signalling in apoptosis (14). In summary, captivating dual function articulate by TGF β signalling wide opens the researchers for a detailed study of its physiology. Regulation of wide variety of physiological functions of a cell is manifested by TGF β signalling pathway. Increased epithelial-mesenchymal transition, increased motility, increased invasiveness, increased colonisation, excess growth stimulation are the hallmarks of TGF β signalling to promote tumorigenesis. In contrast, Growth inhibition, apoptosis, negative angiogenic regulation, maintenance of genomic stability, increased replicative senescence, reduction of immortalization, maintenance of tissue architecture is also governed by TGF β signalling. Several cancer therapeutics are TGF β targeted; specific antibodies are used to check the TGF signalling and observed a high rate of positive result. Inhibition of receptor kinases also gives a promising result in hindering the TGF signalling in tumour cells. The stage from which the TGF signalling turn to tumorigenic is unclear; however, their role in tumour development and progression is highly understood. The implication of RNAi could be an efficient and more effective way to control TGF signalling.
Reference:
1. De Larco, J. E., Preston, Y. A, Cell. Physiol., 09: 143-152, 1981.
2. Anzano, M. A., Roberts, A. B., Lamb, Analytical Biochem., 125: 217-224, 1982.
3. Mario A. Anzano,1 Anita B. Roberts (CANCER RESEARCH 42, 4776-4778, November 1982)
4. Yigong Shi, J Massague (Cell, Vol. 113, 685–700, June 13, 2003) 5. Jack W Penn, Adriaan O Grobbelaar (Int J Burn Trauma 2012;2(1):18-28)
6. Lalage M Wakefield and Anita B Robert (Current Opinion in Genetics & Development 2002, 12:22–29)
7. Frontier in Bioscience (Tang WB, 2010, Jan, 2, 857-60)
8. tumorigenesis Rotraud Wieser, (Current Opinion in Oncology 2001, 13:70–77)
9. Aristidis Moustakas, Katerina Pardali, (Immunology Letters 82 (2002) 85-91)
10. Yin, J.J. et al.(1999). J.Clin.Invest.103, 197–206
11. Ananth, S. et al.(1999) Cancer Res. 59, 2210–2216
12. J. Akhurst and Rik Derynck (Trends in cell biology Vol.11 No.11 November 2001 S44)
13. Xiaohua Yan, Ziying Liu, (Acta Biochim Biophys Sin (2009): 263–272)
14. LC Spender and GJ Inman (Cell Death Differ. 2009 April; 16(4): 593–602
Canonical TGF β signalling, also called Smad signalling involves the Smad (Mothers against decapentaplegic homolog) proteins which transduce the signal to activate a wide range of transcription factors in the nucleus. Eight distinct Smad proteins are identified and are functionally classified into three types; Receptor-regulated Smad (R-Smad), Co-mediator Smad (CoSmad) and the Inhibitory Smad (ISmad). Smad 4 (Co-Smad) is the outcome complex from activated Smad and is forwarded to bring out activation of a transcription factor. Smad6/7 is Inhibitory and competes with R-Smad for the receptor and activates Smurf (Ubiquitin ligase) to degrade activated Smad 4. TGF β can initiate the signal transduction of other pathways independent of Smad called Non-Smad signalling. Different ligands and different receptor belong to the same family can activate the TGF β signalling; each combination of ligand and receptor initiates the cytoplasmic proteins in a different manner and hence results in varied cellular response. Interaction of ligand-receptor is controlled by a special class of proteins called ‘ligand traps’ (4).
Figure 1: involvement of different ligand traps in bringing the ligands to the vicinity of the receptor, Different ligands are associated with specific receptors which in turn activate transducing protein complexes and carries the signal to the nucleus. Smad4 complex is also involved in this signal transduction cascade (4). TGF β signalling is at most important for wound healing, regeneration of damaged tissues and even in scar formation! Signalling process is highly organised and helps in maintenance of homoeostasis. The perhaps controlled inflammatory response is also a direct action formulated by the TGF β signalling cascade (5). Figure 2: Generalised scheme of TGF β signalling cascade. Ligand binding to the receptor dimerizes the cytoplasmic domain of the receptor complex and undergoes phosphorylation. Phosphorylation makes them Serine-Threonine kinase to activate Smad complex by itself becomes dephosphorylated. A hint of Non-Smad signalling is also depicted in the diagram which can also bring a similar cellular response. Smad 7 and Smurf complexes are the negative regulators of TGF β and hence the signalling is set under control (6). SARA (Smad Anchor for Receptor Activation) is a key protein which gathers Smad2 to the cytosolic domain of the receptor; which enhances the possibility for accurate phosphorylation. Epithelial-mesenchymal transition (EMT) can potentially modify the SARA activity (7). In spite of having lifesaving physiological functions, TGF β can even be fatal. Thus, TGF β signalling is designated as a double edged sword as it promotes both pro-oncogenic activation as well as an apoptotic cascade.
Tumour promotion through TGF β signalling: NMA, an orthologue of BAMB protein, blocks the TGF β signalling at an early stage. Overexpression of TGF β signalling and cut down in NMA expression is a clear demarcation in melanoma cells. This is one of the striking examples to illustrate the tumour promotion activity through TGF β signalling. Overexpression of TβRǁ in pancreatic cancer, Hyperactivity of R-Smad in colon cancer justifies the tumour promoting ability of TGF β. Mutations in Smad 2 promote invasiveness rather than blocking the signalling pathway (8). TGF β functions as a growth inhibitor in normal cells; whereas the tumour cells are destined to get the resistance to growth inhibition activity. On the other hand, TGF β secretion would become either autocrine or paracrine mode which would possibly increase the rate of cell proliferation and may even lead to metastasis. Mutations in the protein complexes involved in TGF β deviates the pathway into a tumorigenic. Overexpression of c-myc suppresses TGF β signalling or gives resistance to growth inhibition responses. Along with which c-myc also represses genes of p15 and p21 and thereby results in uncontrolled cell proliferation. The cytokine response may not shut down; in contrast, they exhibit epithelial to mesenchymal transformation which is the initial event in cancer progression and metastasis (9). Crystal clear results were obtained to explain the supportive function of TGF β in metastasis. Breast cancer cells named MDA-MB-231 tend to metastasize to bones. TGF signalling was blocked in these cells and was injected into immunodeficient mice which developed fewer tumours and sailed very fewer osteoclasts (10). Prostate carcinoma cells in immunodeficient mice expressed anomalous angiogenesis by the virtue of overexpressed TGF β signalling. Site-specific administration of antibodies of TGF β neutralised the signalling pathway and reduced the angiogenesis drastically. TGF β presumably activates angiogenesis inducing factor, Vascular Endothelial cell Growth Factor (VEGF) (11). Tumour suppression through TGF β signalling Initial characterization of TGF β signalling revealed the growth inhibition activity as a key cellular response. The cellular architecture was chiefly governed by TGF β signalling and hence could able to maintain the homoeostasis. In normal cells, TGF signalling was found to be very prominent and maintains a threshold level of TGF β. T-->C transition in 29th nucleotide of TGF β enhances the signalling efficiency by increasing its level in serum showed significantly reduced the risk of breast cancer by 50%. Investigation on keratinocyte cell line confirmed the role of TGF β in the maintenance of genomic stability. N-phosphonoacetyl-L-Aspartate treatment to the keratinocyte cells with and without TGF β was assessed and found that the cells with TGF β signalling lesser gene amplification. Restoration of autocrine activity of TGF β lead to drastic reduction in the activity of telomerase. Human colon carcinoma HCT116 cells were characterised after restoration and found that RNA levels of telomerase reverse transcriptase were greatly reduced. Scale down in the activity of telomerase spontaneously drags the cell to senescence. Thus, TGF β plays an indirect physiological role in suppression of cell growth (6). Figure 3: p15 is an effective inhibitor of cyclin-dependent kinases and results in cell cycle arrest. In the absence of TGF β signalling Myc and Miz-1 forms a complex which blocks the transcriptional activation of p15 gene and may lead to tumorigenesis. However, in presence of TGF β signalling, Smad complex disrupts Myc-Miz association and promotes the transcriptome of p15 (12). Smad 6 and Smad 7 are the major Inhibitory Smad which brings negative regulation of TGF β signalling. I-Smad has conserved C-terminal MH2 domain but lacks N-terminal MH1 domain; which are significantly used for phosphorylation in Co-Smad and R-Smad. Single molecule force spectroscopy revealed that even Smad 7 can bind to DNA at Smad Binding Element (SBE) CAGA box. The free N-terminal region of the Smad 7 binds to DNA but not MH2 domain; other Smad complexes bind to DNA with MH2 domain! Hyperactive Smad drastically reduces TGF β signalling and thus results in growth inhibition. On the other hand, Smad 7 activates Smurf1 and 2 to degrade either Smad complexes or ALK5/TβR1 receptor through ubiquitin ligase or proteasomal degradation pathway. Smad 6 functions in different mechanism and it acts as a transcriptional repressor by targeting Hoxc-8 or by binding to DNA or by effective activation of transcriptional corepressor, Histone deacetylase (13). Stronger tumour suppression could be done by forcing the cells to apoptosis; for which TGF β signalling is a highly targeted weapon. Programmed suppression of inhibitors of the intrinsic apoptotic pathway, BCL-X(L), triggers the activation of BIK complex through TGF β signalling. Smad complexes binds to consensus sequence to activate the transcription of BIK; meanwhile it inhibits BLC and sensitises Burkitt’s
Lymphoma (BL) cells to induce apoptosis through TGF β signalling. Thereby it evidently explains the involvement of TGF β signalling in apoptosis (14). In summary, captivating dual function articulate by TGF β signalling wide opens the researchers for a detailed study of its physiology. Regulation of wide variety of physiological functions of a cell is manifested by TGF β signalling pathway. Increased epithelial-mesenchymal transition, increased motility, increased invasiveness, increased colonisation, excess growth stimulation are the hallmarks of TGF β signalling to promote tumorigenesis. In contrast, Growth inhibition, apoptosis, negative angiogenic regulation, maintenance of genomic stability, increased replicative senescence, reduction of immortalization, maintenance of tissue architecture is also governed by TGF β signalling. Several cancer therapeutics are TGF β targeted; specific antibodies are used to check the TGF signalling and observed a high rate of positive result. Inhibition of receptor kinases also gives a promising result in hindering the TGF signalling in tumour cells. The stage from which the TGF signalling turn to tumorigenic is unclear; however, their role in tumour development and progression is highly understood. The implication of RNAi could be an efficient and more effective way to control TGF signalling.
Reference:
1. De Larco, J. E., Preston, Y. A, Cell. Physiol., 09: 143-152, 1981.
2. Anzano, M. A., Roberts, A. B., Lamb, Analytical Biochem., 125: 217-224, 1982.
3. Mario A. Anzano,1 Anita B. Roberts (CANCER RESEARCH 42, 4776-4778, November 1982)
4. Yigong Shi, J Massague (Cell, Vol. 113, 685–700, June 13, 2003) 5. Jack W Penn, Adriaan O Grobbelaar (Int J Burn Trauma 2012;2(1):18-28)
6. Lalage M Wakefield and Anita B Robert (Current Opinion in Genetics & Development 2002, 12:22–29)
7. Frontier in Bioscience (Tang WB, 2010, Jan, 2, 857-60)
8. tumorigenesis Rotraud Wieser, (Current Opinion in Oncology 2001, 13:70–77)
9. Aristidis Moustakas, Katerina Pardali, (Immunology Letters 82 (2002) 85-91)
10. Yin, J.J. et al.(1999). J.Clin.Invest.103, 197–206
11. Ananth, S. et al.(1999) Cancer Res. 59, 2210–2216
12. J. Akhurst and Rik Derynck (Trends in cell biology Vol.11 No.11 November 2001 S44)
13. Xiaohua Yan, Ziying Liu, (Acta Biochim Biophys Sin (2009): 263–272)
14. LC Spender and GJ Inman (Cell Death Differ. 2009 April; 16(4): 593–602
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