CELL SIGNALLING

CELL SIGNALLING

The concept of cell signalling has been progressing for almost decades now. It refers to the communication between the cells and their surroundings. Every single organism, whether unicellular or multicellular, autotrophic, or heterotrophic, depends on cell signalling for its survival. This process takes place by ligand-receptor binding. The ligand binds to the receptor that transduces the required message through controlled reactions. Cell signalling is a crucial step right from cell proliferation to cell senescence. The scope of this essay is to understand the role of growth factors/differentiation factors in the regulation of cell signalling and their significance. It encompasses experimental data providing definite results establishing their importance and clarifying their functioning.

TGF-β FAMILY

TGF-β superfamily is a broad assortment of growth factors including activins, BMP1 , Nodal, TGF-β, and GDNF2 . Activin is mainly produced by placenta, pituitary gland, and major gonads. TGF-β subfamily is composed of three ligands (TGF-β1, TGF-β2 and TGF-β3). TGF-β1 is predominant in lymphoid organs whereas TGF-β2 and TGF-β3 are found in bones and mesenchymal tissues. TGF-β RI and TGF-β RII serve as TGF-β receptors whereas SMAD2 and SMAD3 are substrates for TGF-β family receptors. The TGF-beta signalling is carried out by paired threonine/serine protein kinases (TGFBRI and TGFBRII act as receptors). TGF-β family is responsible for overseeing a range of cell functions, like differentiation, migration, and proliferation. These pleiotropic secreted signalling molecules have unique immunoregulatory properties. Besides that, it also facilitates dorsal-ventral patterning.

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CELL SIGNALLING IN IMMUNE CELLS

Transforming growth factor plays a critical role in maintaining and promoting immune homeostasis, tolerance, and detection of foreign antibodies, and standard functioning of immune cells (like T-cells, helper cells, D cells). It is expressed by both autocrine and paracrine modes to oversee differentiation, proliferation, and activation of the immune cells.

It is considered as the pleiotropic and pivotal regulator of immune responses. Inadequate function of TGF-β cells results in tumour emergence and uncooperative inflammatory responses. Immune-suppression in tumour environment is acutely regulated by TGF- β signalling. To understand the importance of TGF-β signalling, a mouse model (in vivo) with blocked TGF-β signalling in T cells was created (Gorelik and Flavell, 2000). Transgenic mice with dominant negative TGF-β receptor type II (dnTGFβRII) expression and CD4 promoter control were created. This CD4 promoter lacked CD8 silencer which ensured the expression of the transgene in CD8+ and CD4+ T-cells only. Six transgene mice were obtained using DNA microinjection into (C57BL/6xC3H) F1 fertilized oocytes.

Figure 1. CD4-dnTGFβRII Tg+ Mice manifestations

(A) Lung sections display mononuclear cell infiltration in Tg+ mice. Colon sections reveal inflammatory cell deposition in intestinal gland and lamina propria.

(B) Western blotting was done on nuclear protein extract from T-cells from Tg- and Tg+ mice.

(C) Kidney sections from Tg- and Tg+ were stained with FITC-labelled goat anti-mouse IgG.

(Gorelik and Flavell, 2000)

It should be mentioned that the founder mice were identified and backcrossed for additional experimentations. Two Tg+3 pups died suddenly before week 3 and one in week 4. Three remaining mice showed no signs of distress and hence were bred. The progeny was used to confirm the presence of dominant-negative TGF-β receptor type II in the T-cells. Using RT-PCR, presence of dnTGFβRII coding region was confirmed by expression of two spliced mRNA transgenic lines. The progeny continued to live a normal life until 3-4 months when they began showing signs of diarrhoea, sickness, and wasting. Histological examination revealed mononuclear cell infiltration of multiple organs (Figure 1). Infiltration of mucosal propria, sub glandular propria, and muscularis and perivascular subserosa (severe to moderate) with dense infiltration of plasma cells, macrophages, and lymphocytes were observed. Cause of auto-antibody secretion was confirmed by performing western blots (Figure 1) on serum of transgene +positive mice and -negative mice (only Tg+ mice had autoreactive antibodies). Frozen kidney sections were stained with FITC labelled goat anti-mouse IgG. Statistical analysis was done to assess the effect ofTGF-β signalling in T-cells on serum immunoglobulin levels.

This experiment indicated the importance of TGF-β signalling in maintaining T-cell homeostasis. Lack of TGF-β signalling leads to immunopathology, secretion of autoimmune antibodies, and inflammatory infiltration in kidney, lungs, stomach, pancreas, duodenum, and colon (Figure 1). Spontaneous upregulation of MHC class I and II in Tg+ mice might be responsible for abnormal antigen presentation and autoantibody secretion. Furthermore, embryonic lethality of mice (more than 50%) indicated that TGF-β1 plays an important role in embryonic development. It should be noted that TGF-β2 and TGF-β3 were still expressed which might have compensated for TGF-β1. Using transgenic mice with impassable TGF-β signalling, it was concluded that cell mediated multifocal inflammatory lesion, autoantibody secretion, and autoimmune diseases can be induced by absent/lack of TGF-β signalling.

The presence of TGF-β is essential for the maintenance of T-cell homeostasis in a lymphocyte environment. In its absence, T-cells differentiate into effector cells (type I or type II) which result in effector cytokine secretion (such as IL-4 and IFNγ) and increased Tcell dependent immunoglobin levels. It was interesting to note increased expression of serum IgA in CD4- dnTGFβRII mice. To conclude, TGF-β plays an important role in regulating and maintaining T-cell homeostasis and prevents immune inflammation in multiple organs. The mice developed age-related wasting disorder which actively proves TGF-β to be an active anti-differentiation factor.

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CELL SIGNALLING IN EMBRYONIC DEVELOPMENT

During the embryonic development, animal axes formation is orchestrated by signalling centres (called organizers) which oversee developmental events like pattern formation, and cell proliferation. A variety of developmental processes are regulated by TGF-β superfamily, including development of dorsal-ventral body axis (Wu and Hill, 2009).

Lansa and Seaver identified that the TGF-β signalling pathway is responsible for organising cell development in model organism Capitella teleta (Lanza and Seaver, 2018). They used small molecule chemical inhibitors to selectively inhibit BMP pathway or active/Nodal pathway by preventing phosphorylation of type I receptors. Activin/Nodal pathway is activated by ligands TGF-β, Nodal, and Activin whereas BMP2 pathway is activated by ligands BMP5-8, BMP2, BMP4, and ADMP. The expression of the pathways is regulated by antagonists and agonists.

In this study, C.teleta adults were maintained (Seaver et al., 2005) and breeding was done by mating sexually mature males with gravid females (Yamaguchi et al., 2016). Broods were exposed to either DSMO solvent control (controls) or inhibitors (experimental) for three hours. This was done at 4-32 cell stage and 32-256 cell stage respectively (figure 2). Chemical inhibitors SB431542 and DMH1 & dorsomorphin dihydrochloride (figure 2) were used to inhibit Activin/Nodal and BMP pathways respectively. Larval stage was immunolabeled and PCR was used to obtain isolated fragments of the stated4 primers. This was followed by WISH5 and lineage tracing. Lucifer yellow microinjection was used to pressure inject blastomere 2d for imaging.

Specimens were imaged using compound light microscopy and SPOT imaging software. Chi-squared test was conducted to sort the larvae into two categories- (1) all three axes identified, and (2) <3 axes identified.

Figure 2. Effect of DSMO and Dorsomorphin dihydrochloride on embryo.

0.05% DMSO or 5 μM dorsomorphin dihydrochloride were used to treat 6 larvae at specified embryonic stages. Embryos subjected to dorsomorphin dihydrochloride had 4 larval phenotypes: normal (B-B′′), ventrally bent (C′-C′′), partially elongated (D-D′′), non-elongated (E-E′′). Bilateral symmetry, anterior-posterior & dorsal-ventral were observed in 3/4th phenotypes. DIC optics (A-J), Hoechst labelled nuclei (A’-J’), phallidon labelled actin (A′′- E′′ and F′′-J′). (Lanza and Seaver, 2018).

The experiment presented evidence discussing the importance of activin/nodal signalling in specification of trunk identity and retaining a dorsal-ventral axis (4-32 cell stage). SB431542 prevents the phosphorylation of the ALK/4/5/7 receptor which results in the formation of larvae retaining anterior-posterior axis only. During 32-256 cell stage, structural abnormality is observed but all the body axes are detectable and normal head patterning is identified (figure 2).

In annelids, such as Helobdella robusta, BMP5-8 is expressed on the dorsal side against its antagonist gremlin (Kuo and Weisblat, 2011). Surprisingly, BMP absence has no direct effect on axes formation in C. teleta when exposed to the inhibitory drug. Dorsomorphin (BMP chemical inhibitor) clearly affected larval development, but it showed no sign of organizer activity (figure 2). It is intriguing how C. teleta exhibits intense variation in TGF-β signalling by preferring Activin/nodal pathway over BMP pathway.

Hence, the study displays the importance of activin/nodal signalling (via ALK4/5 receptors) as an organising signal. Additionally, the findings displayed the extensive distinctions in spiralian cell development and how TGF- β superfamily controls the axes development and bilateral symmetry formation.

Additional studies might help decipher and confirm the detailed ligands properties utilized in the signalling pathway.

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CONCLUSION

TGF- β super family plays a critical role in an organism’s development and maintenance. It establishes one of the first cues in developing embryo and carries on regulating immune responses in an established system.

Experimental evidences establish that Activin/nodal Pathway and BMP pathway regulate dorsal-ventral axes formation. Experiments on Mice models give us an insight into TGF-β subfamily’s role in maintaining cell homeostasis. TGF-β signalling is important for survival as it regulates basic cell functions like cell proliferation, senescence, cell survival, autophagy, mesoderm induction, inflammatory responses, and immune cell expression, to name a few.

Any alterations/lack in its expression poses a potential threat to the organism’s survival. Ironically, it can be acknowledged that TGF-β serves as a good servant but a bad master.

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