NF- KB (which stands for nuclear factor kappa-light-chain-enhancer of activated B cells, there we called it NF-KB) is a protein complex with multiple functions. It is one of the starting players in the immune system where it stimulates antibodies. It is also important in memory and stress responses. It sometimes can be called as a transcription factor, which control which protein to be produced when you control which DNA is transcribed to RNA and that RNA into protein. We here see the first research paper on NF-KB by Sen, R., and Baltimore, D. (1986) and how they able to find out that NF-KB is inducible in cell lines.

NF-KB is a nuclear protein that interacts with a defined site in the k immunoglobulin enhancer. 

INTRODUCTION

With the ability to reintroduce genes into cells has allowed the definition of regulatory sequences, a segment of a nucleic acid molecule that is capable of increasing or decreasing the expression of a specific gene, a novel regulatory element known as enhancers.  Enhancers were originally seen in viral genomes and inducible genes. There are many levels of regulation – Positive, Negative and Inducible – mediated by enhancer sequences and are important in study gene expression. 

As a step towards understanding the mechanism of tissue-specific enhancer function, scientists at that time introduce five factors that interact with the B-cell-specific heavy chain and k light chain enhancers.

The most interesting of these factors, NF-KB, is one that interacts only with k enhancer and seems to be step specific within the lymphoid line,

  • Expressed in mature B cells and plasma cells 
  • NOT in pre-B cells or T cells.

This corresponds to those cell types in which k transcription is normally active which shows this factor may be necessary for k gene expression.

Scientists taken a cell line contains only pre-B cell through transcriptionally inactive k locus. Cells in this cell line may be undergoing a transition from a pre-B to B cell by stimulated by B cell mitogen bacterial lipopolysaccharide (LPS), during which k transcription is initiated, k protein is synthesized and assembled IgM molecule is transported to the cell surface. 

THE ROLE OF NF-KB AS A CRITICAL ACTIVATOR

Role of NF-KB is a critical activator of k enhancer that require its inducibility in pre-B cell lines after an appropriate stimulus.                                                                                                                                                                                                                                                                                                                

  1. NF-KB can be induced in pre-B cell lines by LPS

 Pre-B cell line stimulated with LPS for 20 min to check whether NF-KB inducible in this cell line. And nuclear extracts derived from these cells’ presence of NF-KB can be found by electrophoretic mobility shift assay. To assay for NF-KB a DNA fragment containing binding site fragment was labeled and incubated with unstimulated cell extracts lacked a major band with B cell extracts. And stimulated cells show nucleoprotein complex band. This experiment done on other cell lines also and show the same result. 

EXPERIMENT ON CELL LINES

Fig: Show Electrophoretic mobility shift assay on cell line and show B cell bands

Characterized this band by competition experiments and find the binding site by

  • By methylation interference experiment. In this band produced by the interaction of the NF-KB factor with the B site within the k enhancer (a site containing the sequence GGGGACTTTCC)

Thus, two pre-B cell lines are clearly induced by LPS for NF-KB activity.

2. Induction of NF-KB by LPS Does not require Protein synthesis

  • Lines induction of k gene expression was obvious in the cells which are pretreated with translation inhibitor cycloheximide or anisomycin.
  • Uninduced cells in cell line were negative for NF-KB
  • And treatment this cell line with LPS alone give NF-KB positive test
  • Treatment with cycloheximide or anisomycin alone also give a positive test
  • Treatment with both LPS and cycloheximide will give you the super-induction of NF-KB.
  • Treatment with both anisomycin and cycloheximide will give less superinduction of NF-KB.

Treatment of cells with cycloheximide alone or LPS alone gave an approximately equivalent amount of NF-KB in the cell extract.  But the anisomycin alone appeared to be much lower. This can be because of drug toxicity because after treatment with anisomycin cells looks unhealthy. 

factors which induce NF-KB
fig: flow chart showing how LPS and translation inhibitors affect NF-KB level

3. Phorbol ester can induce NF-KB in the cell line

The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), has shown to induce surface immunoglobin in the cell line. Presumably via activation k transcription and transport of immunoglobulin to the cell surface. 

  • And thus, active phorbol ester by itself is capable of inducing NF-KB possibly by protein kinase C as a post-translational modification.
  • An inactive phorbol ester (phorbol12,13- didecanoate, 4alpha-DPP,) did not cause induction of NF-KB under similar conditions
how phorbol ester affect NF-KB level
Fig: how phorbol ester induce NF-KB in the cell line 

INTERPRETATION

Scientists have studied the induction of nuclear factor NF-KB that can be detected in crude nuclear extracts by EMSA. NF-KB interacts with a B site in the k immunoglobulin gene enhancer and its presence in B cell and plasma cell. 

  • And current analysis shows us that NF-KB induction in the cell can be induced by mitogen LPS in two cell lines representing the pre-B stage of B cell differentiation.
  • Induction of this factor involves a post-translational modification of a preexisting protein. And this proves by induction in the cell even take place in the presence of translation inhibitors like cycloheximide and anisomycin. Scientist believes that this inhibitor binds to the k locus thus preventing transcription of the gene. This inhibitor probably interacts with NF-KB itself, not with the chromosomal DNA.
  • These translational inhibitors in coaction with LPS to produce a super-induction. Then this means there should be a direct activation pathway
  • An active phorbol ester like PMA can induce NF-KB by itself and the time course of this activation is more than LPS alone. 
  • This is because LPS binds to a receptor that activates phospholipase C, which activates inositol trisphosphate and diacylglycerol, being the physiological activator of protein kinase C. whereas PMA directly acts on protein kinase C, therefore PMA give a faster response

Previously scientists know that restricted tissue arrangement of NF-KB and enhancer make it possible that B-cell-specific expression of k genes. So, from what discussed above make it even stronger because various stimuli that activate k transcription in pre-B cell lines show parallel effects on NF-KB activity. 

  • Results of LPS treatment provide the strongest argument as LPS both induces k gene transcription and induces NF-KB. 
  • As anisomycin was found not to induce k transcription in the pre-B cell so scientists say treatment with the toxic drug for long term causes inhibition of protein synthesis.

The aim of this paper is that cell-specific enhancer-binding protein may be induced by the post-translational modification of a pre-existing protein. 

For the expression of k gene expression, the pre-existence of an NF-KB precursor brings the regulator immediate stop of the regression chain. The precursor to NF-KB is presented in pre-B cells. And it suggests expression when a cell responds to either internal or external signals during ontogeny. And NF-KB is also present in viral enhancers (SV40), in HIV.

Scientists know that NF-KB protein and B site are used for a specific purpose during B lymphocyte differentiation. In this post-translational activation event, with the involvement of protein kinase C (PKC) is the phosphorylation of pre-NF-KB. In this labile repressor can be a phosphatase or a molecule that blocks the site of phosphorylation by direct interaction with pre-NF-KB. And such inhibitors are not B-cell-specific this suggests that stabilization of NF-KB in B cells is a consequence of inaccessibility to the NF-KB inhibitor.

So, induction of NF-KB appears to involve a posttranslational modification mediated either directly or indirectly by protein kinase C, which is itself a cell signal. scientist results imply that tissue-specific posttranslational modifications may generate factors that regulate gene expression, and thus the data bear on the general problem of activation of tissue-specific genes. NF-KB as it involves multiple cell signaling pathways and also seen to take part in cancer regulation.

REFERENCE 

Sen, R. and Baltimore, D., 1986. Inducibility of κ immunoglobulin enhancer-binding protein NF-κB by a posttranslational mechanism. Cell47(6), pp.921-928.

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