The Effect of Platelet Derived Growth Factors and Lipopolysaccharrides on KB cells Proliferation.

Sobukonla Timothy and Karen Louis

ABSTRACT
Keratinocytes are stratified, squamous, epithelial cells that make up 95% of the cells in the epidermis. These cells are found in the basal layer of the stratified epithelium of the epidermis, and are to as basal cells, or basal keratinocytes. Keratinocytes play a vital role in the structural component of the skin, as well as a vital function in immune response. Platelet derived growth factor is a powerful mitogen for many cell types such with keratinocytes growth and motility. Lippolysaccharide (LPS) is found on the cell surface of gram-negative bacteria and assist in the stabilization of the overall cell wall structure. LPS along with CD14 and Toll-like receptor 4 expressed on the cell surface of keratinocytes, play a vital role in regulating the innate immune response system and influencing the adaptive immune response mechanism.
INTRODUCTION
Keratinocyte cells are stratified, squamous, epithelial cells that comprise of skin and mucosa, including oral, esophageal, and genital epithelia. 95% of the cells in the epidermis are keratinocytes. Keratinocytes are tightly bonded together to form a strong layer between the nerves of the skin and underlying tissues of the epidermis. These cells originate in basal layer of the stratified epithelium and are referred to as basal cells or basal keratinocytes. In addition to their structural role of preserving the health and integrity of the skin, basal keratinocytes play a vital function in immune response. The skin is the first organ in which pathogens encounter. Keratinocytes form a protective barrier that prevents the entry of any foreign and toxic substances from the environment through the skin into the body as well as avoiding the loss of moisture, heat, and any other essential elements of the body. Keratinocytes also serve as immunomodulaters, responsible for secreting inhibitory cytokines in the presence of a skin breach, stimulating inflammation and activating Langerhans cells. Langerhans cells are found in the skin and serve as antigen-presenting cells. These cells are the first to process microbial antigens entering the body from a skin abrasion. Keratinocytes first reside as keratinocyte stem cells in the basal layer of the epidermis, the lowest layer of the stratified epithelia. These cells have a low mitotic rate and divide to produce transient amplifying cells that further divide and differentiate as they move upwards in the epidermis. These cells are found above the spinous layer (thickest layer of the epidermis). The differentiating cells produce compounds and other proteins which are critical to the integrity of the outermost layer of the skin, the stratum corneum. The keratinocytes in this layer dead squamous cells that no longer multiplying or dividing. Once keratinocytes reach the corneum, they are keratinized, or cornified, creating the tough outer layer of skin. The most common type of skin cell protein found inside keratinocytes are keratins. Keratins are a filamentous protein that form the cytoskeleton of keratinocytes and is the primary component of and nails. Defects in keratin expression result in various diseases of the epidermis, as well as the hair and nail problems. Keratinocyte cells are continuously being discarded and replaced from the outer most layer of the skin. In this regard, keratinocytes can be studied to investigate various skin cancers that disrupt this process. Most skin diseases or cutaneous lesions described by epidermal hyperplasia or hyperkeratosis are due to a decrease in keratinocyte apoptosis. In psoriasis, a chronic inflammatory skin disease, there is decreased spontaneous keratinocyte apoptosis in lesional skin. This effect is associated with decreased levels of caspase-14 .Caspase-14, is a protein involved in apoptosis. Caspase-14 is activated during the last stage of skin cell maturation. Consequently, caspase-14 leads to the outer epidermis of the skin containing dead cells that are discharged as flakes of skin. The absence of caspase-14 has very harmful consequences on the skin's protective barrier function, resulting in a loss of water and decreased protection against UV light. Squamous cell carcinoma, leads to activation of keratinocyte survival signaling pathways. Activation of epidermal growth factor receptor (EGFR) activates the transcription Stat3 axis (Deepak et al, 2006) which in turn prompts antiapoptic proteins such as Bcl-xL and Mcl-1. The identification of molecules involved in caspase-14 dependent processes is of primary concern in pharmaceutical industries, which are continuously seeking new methods to avert sunburn and ageing of the skin. Pomegranate extracts have been found to supply protection against UV damage of keratinocytes and is actively included in skin care products such as wrinkle-reducing creams. In this study, KB cells were used which contain keratins that are found in keratinocytes to study the behavior of the cells in relation to cell proliferation. This was investigated by using two stimulants, Platelet Derived Growth Factor (PDGF) and Lipopolysaccharide (LPS). Platelet derived growth factor (PDGF) is made up of either a homo or heterodimeric chains A and B. This growth factor is a powerful mitogen for many cell types such as keratinocytes, macrophages, vascular endothelium and fibroblasts. PDGF binds to two non-identical transmembrane tyrosine kinase receptors, causing dimerization and autophosphorylation of these receptors. This in turn, creates a docking site for adaptor proteins such as Src homology 2 (SHC) domain containing signaling molecules, and activating a downstream of several signaling pathways. PDGF is localized in the α- granules of platelets and are released upon a skin breach. This initiates chemotaxis of neutrophils, fibroblasts and smooth muscle cells to the site of injury. Macrophages are also stimulated and cause the production and release of growth factors such as transforming growth factor beta (TGF-β). PDGF plays an important role in blood vessel maturation. In vivo experiments demonstrated that this growth factor is important in recruiting pericytes, (connective tissue cells) to the capillaries and thus increase the structural integrity of these vessels (Barrientos et al, 2008). PDFG functions as to stimulate reepithelization by up regulating the production of IGF-1 (Insulin Growth Factor-1) and thrombospondin -1. IGF-1 has been shown to increase keratinocyte motility along with growth. Thrombospondin-1 is a protein found in humans that is programmed by the TSP1-gene. Thrombospondin 1 (TSP1) inhibits angiogenesis and alters endothelial cell linkages, motility, and growth by delaying cell degradation resulting in a proliferative response. Lipopolysaccharide (LPS), or lipoglycans, are large molecules consisting of a lipid portion and a polysaccharide segment joined by a covalent bond. LPS is found in the outer membrane of most gram-negative bacteria. LPS increases the negative charge of the cell membrane and assist in the stabilization of the complete cell wall structure. It is known as an immunogenic antigen and functions by enhancing immune response. Toll-like receptors (TLR’s) are transmembrane proteins that consist of extracellular domains of leucine-rich repeats and a cytoplasmic region similar to that of IL-1 receptor (Peter et al, 2002). TLR’s contributed to a multitude of inflammatory responses and perform vital role in innate immune response along with stimulating adaptive immunity. Keratinocytes express CD14 and TLR 4 on their cell surface. LPS-binding protein facilitates the binding of CD14 molecule and TLR4 to form a CD14- TLR4 complex. This complex stimulates the rapid release of intracellular calcium and tyrosine kinase phosphorylation. The intracellular domain of the TLR activates a MyD88-dependent pathway that ultimately leads to the nuclear translocation of nuclear transcription factor NF ҝB. NF ҝB functions to modulate the expression of many immune response genes and the release of proinflammatory cytokines and chemokines such as alpha tumor necrosis factor (α-TNF), IL-6, and IL-8 (Jamie et al, 2004).
MATERIALS AND METHODS
Cell culture: Cells were grown in media containing DMEM, Fetal bovine serum, glutamin, penicillin/streptomycin solution and brought to a final concentration of 10% serum in a cell culture flask placed in a 370 CO2 incubator. The cells used are KB cells derived from epidermal carcinoma of the mouth and are adherent cells. The cells were split every two to four days to avoid apoptosis. This process was continued until the cells were needed for experimenting.
Cell count: The first thing we did was count our cells using a heamocytometer and a microscope. We counted our cells by adding trypan blue dye to already diluted cells and counted the amount of cells we could see through the microscope. We used this to get an estimate of the amount of cells we have available per ml and diluted or brought the concentration to 1x106.
Proliferation assay: We set up a proliferation assay by firstly plating our cells in a 96-well plate. This was done by adding medium to well A, cells were added to wells B-H, this was done in triplicate by adding three sets of each in order to have a more accurate data. The plates were incubated at 370C for 24 hours before adding our stimulants to cells. The PDGF (platelet derived growth factor) stimulants were added at 10ng,100ng and 1000ng concentrations to well C,D,E respectively and the LPS (Lipopolysaccharrides) were added to the wells F, G and H at 5ng, 50ng and 500ng concentrations. This was then incubated for 24hrs or 48hrs depending on timeline before being read.
Cell tither assay: In order to read our cells, we setup a cell tither assay. We added 20µl of MTS to all our wells which will break our cells down into formazan which is read to determine proliferation. The cells were incubated for 2-3 hours before being read at absorption of 490nm in a 96-well microplate reader. Our results were then analyzed by using Microsoft Excel software to create analytic Bar charts comparing our cells to stimulated cells and identifying the effects of the stimulants on the cell proliferation.
Timeline and serum starvation: we also used different parameters to see how well our cells responded to proliferation. We reduced our cell concentration to 5% and 2.5% from the normal 10% by a process called serum-starvation where the amount of fetal bovine serum is reduced significantly to change how the cells will normally grow. Another thing we did was compare two different stimulation timeline in comparing response of our cells to growth factors over both 24 and 48 hours time.
RESULTS
After performing all our experiments we used Microsoft excel software to create Bar charts to determine proliferation of KB cells. We noticed that when 10% serum concentrated (normal) cells were treated with PDGF; there was an increase in the proliferation rate of our cells, although the lower the PDGF concentration the more proliferation observed in each case (fig 1A). When the 10% serum concentrated cells were treated with LPS at the same 24 hours timeline, we found out our cells proliferated when treated with 5ng LPS and 50ng LPS, but at 500ng of LPS our cells growth was highly inhibited (fig 1B). When our cells were serum starved to 5% serum concentration and treated with LPS and PDGF for 24 hours, our cells showed proliferation at all concentrations for PDGF but increased with low concentration of the growth factor (fig 2A). LPS showed the same trend with the 10% serum concentrated cells, as proliferation occurred at 5ng and 50ng, but was inhibited at 500ng (fig 2B). At 2.5% serum-starvation after treatment with Lipopolysaccharrides and platelet derived growth factor for 24 hours, there was a change in the proliferation trend of our cells as in the case of PDGF, proliferation occurred at 10ng and 100ng concentration and inhibition of cell growth at 1000ng like in LPS for 5% and 10% serum concentration (fig 3A). LPS on the other hand showed proliferation at all concentrations even though cell growth reduced by concentration (fig 3B). When our ten percent serum concentrated cells were treated with PDGF and LPS at a changed timeline of 48 hours, we noticed a significant change in the proliferation rate of PDGF as 10ng concentration led to cell growth inhibition, 100ng showed no significant change and proliferation occurred at 1000ng (fig 4A). LPS treatment at 5ng, there was slight proliferation, no change at 50ng and inhibition or reduced cell proliferation at 500ng (fig 4B). For our 5% serum starved cells treated with LPS and PDGF for 48 hours, we noticed slight increase in proliferation rate at 5ng and 50ng and reduced growth at 500ng for LPS (fig 5B). PDGF treated cells show proliferation increase with increase in the concentration of the growth factor.
DISCUSSION
Proliferation which simply put means cell growth was measured in KB cells. It was shown that proliferation occurred for most of our cells after being treated at different stimulations of PDGF and LPS at different timelines but there were also cases of reduced growth of cells. We noticed that for both 10% serum concentrated and 5% serum-starved cells treated with LPS and PDGF for 24 hours, the same proliferation pattern was observed telling us that when the KB cells are serum starved to about 5%, there was no change in the manner in which they proliferate and act, but at 2.5% serum starvation and stimulation with the same growth factors for the same timeline of 24 hours, there were changes in the way the cells reacted to the stimulants as proliferation that was normally increased at 1000ng PDGF was shown to be reduced and also LPS showed proliferation at all concentrations of LPS, from all these, we deduced that this might be a result of the cells growing slower because of the reduced Fetal Bovine serum, which can lead to increased reaction to the higher stimulant concentrations as opposed to 5% and 10% serum concentration. When we changed the time of stimulation form 24 hours to 48 hours, we noticed that our cells reacted differently to the stimulants for our 10% serum concentrated and 5% serum-starved cells. For the 10% serum concentrated cells, proliferation increased with increased PDGF level which was different from the cells at 24 hour, as there was reduced growth with 10ng concentration, no change at 100ng concentration and proliferation at 1000ng, we suggested that this may be the result of increased receptor level or even decreased level with the amount of time the stimulation occurred making the most concentrated PDGF have proliferation and lower level to cause inhibition of cell growth. LPS in this case showed similar trend with the 24 hour timeline although the proliferation levels were slightly lower than normal. For the 5% serum starvation, we saw the same trend as the 10% serum-concentration after 48 hour, although proliferation occurred at all the concentration level showing once again that reduced serum level can be effective in explaining how proliferation works in KB cells. LPS was the same with the 10% serum concentrated cell after 48 hours showing that the LPS are much more constant in the way we saw them affect our cells except at 2.5% serum-starvation which might be the result of overly reduced serum level. We found out that both growth factors were effective in making our cells proliferate, although at different timelines during our cell growth they acted differently showing that the growth of our cells within that time and the cell cycle can affect proliferation. For future studies we would like to look at the cells closely to see how the growth factors bind to the receptors and also note if there are change in receptor level and reaction at different timelines in our cell. Overall we were better educated about cell signaling in relation to proliferation and cell survival after doing this experiment.

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