Cell Signaling

1. Cell Signaling Lecture 12

2. NFkB • NFkB (nuclear factor kappa beta) is a transcription factor that plays important roles in the immune system. • NFkB regulates the expression of cytokines, inducible nitric oxide synthase (iNOS)-the enzyme that produces nitric oxide which is toxic to bacterial cells. • inhibitors of apoptosis protein that prevent cell death and effector enzymes in response to ligation of many receptors involved in immunity including T-cell receptors (TCRs), B-cell receptors (BCRs) and members of the Toll-like receptor/IL-1 receptor super family. • NFkB also plays a role in the development and the activity of a number of tissues including the central nervous system, liver cells etc. • Moreover, pathological dysregulation of NFkB is linked to inflammatory and autoimmune diseases as well as cancer.

3. NFkB family • In mammals, the NFkB family is composed of five related transcription factors: p50, p52, RelA (p65), c-Rel and RelB. • These transcription factors are related through an N-terminal, 300 amino acid, DNA binding/dimerization domain, called the Rel homology domain (RHD), through which they can form homodimers and heterodimers that bind to 9-10 base pair DNA sites, known as kB sites, in the promoters and enhancer regions of genes, thereby modulating gene expression. • RelA, c-Rel and RelB contain C-terminal transcriptional activation domains (TADs), which enable them to activate target gene expression. In contrast, p50 and p52 do not contain C-terminal TADs; therefore, p50 and p52homodimers repress transcription unless they are bound to a protein containing a TAD, such as RelA, c-Rel or RelB or Bcl-3 (a related transcriptional co-activator)..

4. NFkB’s transcriptional activity is silenced by interactions with inhibitory IkB proteins present in the cytoplasm. • There are currently seven identified IkB family members - IkBa, IkBb, Bcl-3, IkBe, IkBg and the precursor proteins p100and p105

5. NFkB signaling pathway

6. There are two signaling pathways leading to the activation of NFkB known as the canonical pathway (or classical) and the non-canonical pathway (or alternative pathway). • The common regulatory step in both of these cascades is activation of an IkBkinase (IKK) complex consisting of catalytic kinase subunits (IKKa and/or IKKb) and the regulatory non-enzymatic scaffold protein NEMO (NF-kappa B essential modulator also known as IKKg). • Activation of NFkBdimers is due to IKK-mediated phosphorylation-induced proteasomal degradation of the IkB inhibitor enabling the active NFkBtranscription factor subunits to translocate to the nucleus and induce target gene expression.  • NFkB activation leads to the expression of the IkBa gene, which consequently sequesters NFkB subunits and terminates transcriptional activity unless a persistent activation signal is present.

7. Conical/classical • In the canonical signaling pathway, binding of ligand to a cell surface receptor such as a member of the the Toll-like receptor superfamily leads to the recruitment of adaptors (such as TRAF) to the cytoplasmic domain of the receptor. • These adaptors in turn recruit the IKK complex which leads to phosphorylation and degradation of the IkB inhibitor. • The canonical pathway activatesNFkBdimers comprising of RelA, c-Rel, RelB and p50.

8. Canonical and non-canonical NF-κB signaling pathways. Canonical pathway is triggered by numerous signals, including those mediated by innate and adaptive immune receptors. It involves activation of IKK complex by Tak1, IKK-mediated IκBαphosphorylation, and subsequent degradation, resulting in rapid and transient nuclear translocation of the prototypical NF-κBheterodimerRelA/p50. Non-canonical NF-κB pathway relies on phosphorylation-induced p100 processing, which is triggered by signaling from a subset of TNFR members. This pathway is dependent on NIK and IKKα, but not on the trimeric IKK complex, and mediates the persistent activation of RelB/p52 complex.

9. Non-conical • The non-canonical pathway is responsible for the activation of p100/RelB complexes and occurs during the development of lymphoid organs responsible for the generation of B and T lymphocytes. • Only a small number of stimuli are known to activate NFkB via this pathway and these factors include lymphotoxin B and B cell activating factor (BAFF). • This pathway utilizes an IKK complex that comprises two IKKa subunits, but not NEMO. In the non-canonical pathway, ligand induced activation results in the activation of NFkB-inducing kinase (NIK), which phosphorylates and activates the IKKa complex, which in turn phosphorylates p100 leading to the processing and liberation of the p52/RelB active heterodimer. In contrast to p100, p105 undergoes constitutive cleavage to producep50, whether p105 can undergo inducible processing remains a contentious issue.

10. Role of NFkB in diseases • Asthma is a chronic inflammatory disorder. The pathogenesis of asthma involves the persistent expression of pro-inflammatory cytokines, chemokines and other such inflammatory mediators. Many of these genes contain the kB site for NFkB within their promoters, suggesting that NFkB plays a vital role in asthma. Indeed, increased NFkB activity has been observed in the airways of asthmatic patients. • NFkB is also implicated in inflammatory bowel disease such as Crohn’s disease and ulcerative colitis. • NFkBactivationis evident in biopsies from such patients and treatment of patients with steroids decreases NFkB activity in biopsies as well as reducing the clinical symptoms of disease. Furthermore, NFkB is involved in the pathophysiology of the autoimmune disorder rheumatoid arthritis (RA). NFkB itself is upregulated in RA and cytokines such as TNFa that activate NFkB are elevated in the synovial fluid of patients with RA. • In addition to the roles that NFkB plays in inflammatory diseases, constitutive activation of the NFkB pathway is involved in some forms of cancer such as leukemia, lymphoma, colon cancer and ovarian cancer. •   Mutations that can lead to such tumors include those that inactivate IkB proteins as well as amplifications and rearrangements of genes encoding the NFkBtranscription factor subunits. However, more commonly it is thought that changes in the upstream pathways that lead to NFkBactivation become deregulated in cancer.

11. Assignment topics • G1: Herdgehog Signaling pathway in breast cancer development • G2:RTK/RasMAP signaling pathway • G3: Notch signaling pathway • G4: PI3K/AKT pathway • G5: EGFR and Ras Signaling pathway • G6: Ras/Raf/MEK/ERK • G7: NFkB signaling pathway • G8: JAK/STAT signaling pathway

12. Assignment should include • What is breast cancer? • Types of breast cancer • Stages of breast cancer • Epidemeology of this cancer world wide, South East Asia and Pakistan. Write in tabulated form • Introduce and explain your signaling pathway with schematic diagram or cycle. • What happens in breast cancer when this pathway gets disrupted and how? Include diagram or cycle • Potential treatments for making the pathway normal. • References with every information is required otherwise you will lose marks • Pages 10. • DEADline: 15 May

13. Table of statistics For the American and European data, you should give the reference of a paper or data by US cancer research or UK cancer research For South East Asian and Pakistan data, include references from WHO, ShoukatKhannam or research papers.