Role of functional Toll like receptor (TLR) 2/6/1 heterodimers in regulated and unregulated growth of HEK293 cells

Zoe Little, Jane Mitchell & Mark Paul-Clark, Imperial College London.

Toll like receptors (TLRs) are pattern recognition receptors (PRRs) which sense pathogen associated molecular patterns (PAMPs). It is increasingly recognised that TLRs are central to innate immune responses as well as contributing to some forms of inflammation (Mitchell et al., 2007). However, the role of TLRs in homeostatic processes is only recently being investigated. In the current study we have used human embryonic kidney cells (HEK293) transfected with TLR2, TLR2/1, TLR2/6 or a null vector to investigate the role of these receptors in cell proliferation.

HEK-TLR2, HEK-TLR2/1 or HEK-TLR2/6 were cultured in DMEM according to supplier’s (Invivogen, USA) instructions. Cells were plated at 30 x 103 or 100 x 103 cells per well in 96-well plates and incubated over night in DMEM containing 10% fetal calf serum (FCS). Medium was then replaced with fresh DMEM with or without 10% FCS. Cells were incubated in an atmosphere of 5%CO2at 37oC for 48 hours before being counted using a haemocytometer and a light microscope at x40 magnification.

Figure 1: Cell replication patterns in HEK-TLR2, HEK-TLR2/1 or HEK-TLR2/6 cells stimulated with or without serum. The data represents the mean ± S.E.M. for n=3. Differences between the number of cells seeded and after 48 hours were tested by two-way ANOVA. Where P<0.05 is denoted by ***.

As expected, all cells proliferated in the presence of FCS. However, more cells were present in HEK-TLR2 than HEK-TLR2/1 or HEK-TLR2/6 cultures after stimulation. Moreover, HEK-TLR2 cells proliferated significantly in the absence of serum. By contrast, HEK-TLR2/1 or HEK-TLR2/6 did not proliferate in DMEM without FCS. TLR2 requires dimerization with TLR1 or TLR6 in order to function. In the current study we found that where TLR2 is expressed alone cells displayed an ‘aberrant’ growth pattern, by proliferating in the absence of added serum. These observations suggest that the presence of a functional TLR2 heterodimer prevents intrinsic division in these cells, which may have implications for our understanding of proliferative inflammation such as that associated with cancer.

Mitchell J.A. et al. (2007). J Endocrinol, 193, 323-30.

This work was funded by the British Lung Foundation.