NIH3T3 fibroblasts respond to TNFα-mediated signaling in a geometry-dependent manner, which is essential for maintaining their cellular homeostasis. In cells of different geometries (circular vs rectangular), TNFα induces differential translocation of transcription factors and the expression of different set of target genes.
This study was published in PNAS in 2017.
Mitra A et al. cell geometry dictates TNFα-induced genome response. 2017. PNAS. 114(20): E3883-3891. doi: 10.1073/pnas.1618007114.
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Figure: NIH3T3 cells respond to TNFα signaling in a geometry-dependent manner. In rectangular cells, TNFα induces the nuclear localization of transcription factor NFκB, while causing another transcription factor MKL to exit the nucleus. In circular cells too, TNFα induces the nuclear localization of NFκB, whereas MKL, which is mostly cytoplasmic, retain their subcellular localization. Based on the type and magnitude of transcription factors localizing in the nucleus, different gene expression programs are activated in NIH3T3 cells in a geometry-dependent manner.
This study describes a close interplay between the mechanical state of the cell and the biochemical signals arising from its surroundings in regulating its nuclear signaling and gene expression programs. By using fibronectin micropatterns to constrain NIH3T3 fibroblasts into rectangular or a circular geometries, the study shows how cells of different geometries respond to TNFα-mediated signaling through differential localization of transcription factors and modulation of their gene expression programs. For instance, in rectangular cells, TNFα-mediated signaling induces Rho inhibition and F-actin depolymerization that leads to the nuclear shuttling of the transcription factor NFκB. TNFα-induced F-actin depolymerization also causes another transcription factor, MKL (it primarily associates with G-actin), to exit from the nucleus and translocate to the cytoplasm. However, in circular cells- which have high NFκB and low MKL in their nuclei to begin with- TNFalpha treatment does not lead to significant changes in the localization of these transcription factors. Therefore, TNFalpha signaling defines distinct genetic programs for different mechanical states of the cell. This is important for cells to maintain homeostasis within the constantly evolving tissue environment.
Understanding the basics
Cells are capable of relaying mechanical stimuli from their physical environment all the way down to the nucleus through electrochemical, biochemical or mechanical pathways. In many cases the activation or suppression of a given pathway by a mechanical cue gives rise to alterations in gene expression. Mechanical signals initiate biochemical pathways through conformational changes in mechanosenstitive proteins that leads to chemical signal propagation and amplification. These proteins then interact with downstream mediators before eventually reaching the nucleus and initiating changes in gene expression. This final ‘end point’ in the pathway can be achieved through activation of transcription factors, the formation of transcription complexes or through a physical remodeling of chromatin or other nuclear components. Of particular importance in biochemical mechanotransduction are phosphorylation events or other post-translational modifications of proteins, as these events are often associated with changes in transcription. Furthermore, mechanosensitive proteins may also mediate the nuclear translocation of transcriptional regulators, where they bind to specific regulatory sites on DNA to initiate gene expression.
NFκB (nuclear factor kappa light chain enhancer of activated B cells) is a family of highly conserved transcription factors that regulate many important cellular behaviours, in particular, inflammatory responses, cellular growth and apoptosis. NFκB is also involved in diseases such as cancer, arthritis and asthma. NFκB is formed through the homo- or hetero-dimerization of members of the Rel family of DNA binding proteins. In mammals these include RelA (p65), c-rel, RelB, p105 (the precursor of p50), and p100 (the precursor of p52). Each member of this family contains a Rel homology domain (RHD), which itself consists of a DNA binding region, a dimerization region and a nuclear localization signal. NF-κB activity is regulated by family of proteins known as IκBs which include IκBα, IκBβ, IκBγ, IκBε and Bcl-3. When in their inactivated state, NFκB complexes are localized in the cytoplasm, in complex with IκB kinase-α (IKKα), IκB kinase-β (IKKβ) and IKKγ/NEMO, a non-enzymatic protein that may function as a scaffold. The IKK complex can be activated by various cytokines, inflammatory molecules and stress signals. Depending on the composition of NFκB in the nucleus, different genes will be actively transcribed. This variation and specificity in gene targets is further determined by the post translational modification of different subunits, such as the phosphorylation of p65. NFκB has also been identified as a mediator of mechanotransduction in several cell types. This role is carried out through changes in both its activation, and localization, in response to mechanical signals.
Actin filament depolymerization helps to maintain a pool of actin monomers that permits the continual restructuring and growth of the actin cytoskeleton. Disassembly of actin filaments occurs at the pointed end of the filament and is driven by the ADF/cofilin (AC) family of proteins. Actin monomers intrinsically dissociate from the barbed end at a faster rate than they do from the pointed end. This is counteracted by the binding of capping proteins or formins to the barbed end, creating a more stable filament. The action of cofilin at the pointed end serves to destabilize the filament and promote the release of ADP-actin monomers. The destabilized form of actin filaments, which has been compared to that observed in younger filaments, is more prone to filament severing. The higher affinity of ACs for ADP-F-actin relative to ATP-F-actin causes severing in the central regions of filaments where ADP-actin is enriched, though depolymerization at the pointed end also occurs. The phosphorylation of cofilin greatly reduces its F-actin binding and depolymerizing activity. Cofilin phosphorylation in vertebrates is controlled by the activity of Rho GTPase and Lim kinase pathways.
The Study in Detail
- The impact of mechanical signals such as cell geometry on cellular response to biochemical signals was tested by constraining the growth of NIH3T3 fibroblasts on two different micropatterns- a rectangular, anisotropic pattern and a circular, isotropic pattern.
- TNFα treatment led to increased nuclear translocation of p65 in circular cells compared to rectangular cells.
- In the absence of TNFα stimulation, the cytoplasmic levels of IkB (inhibitor of p65 nuclear translocation) were higher in rectangular NIH3T3 cells than in circular cells. TNFα stimulation led to increased phosphorylation of cytoplasmic IkB and its rapid degradation in rectangular cells.
- The cytoplasmic levels of F-actin and phospho-Myosin Light Chain (pMLC) were higher in rectangular cells than in circular cells. The levels of phospho-Lim kinase-2 and phospho-cofilin (inactive form that cannot depolymerize F-actin) were also higher in rectangular cells. The findings suggested a higher Rho GTPase activity in rectangular cells.
- TNFα treatment led to a decrease in Rho activity in rectangular cells. There was a decrease in phospho-Lim kinase-2 and phospho-cofilin levels, and an increase in actin filament severing by “active” cofilin, resulting in significantly lower F-actin intensities. In circular cells, F-actin intensities (low to begin with) did not change drastically.
- In rectangular cells treated with an F-actin polymerization inhibitor- either alone or in combination with TNFalpha- degradation of cytoplasmic IkB and nuclear translocation of p65 increased. This suggested an important role for actin depolymerization in triggering NFκB pathway in cells.
- In TNFα-stimulated rectangular cells, F-actin depolymerization occurred due to a loss in Rho activity. This was evidenced by a significant drop in IkB degradation and p65 nuclear translocation in the presence of a constitutively active form of Rho (RhoV14).
- In unconstrained NIH3T3 cells, TNFα treatment caused the depolymerization of F-actin into structures called actin nodes, which function as scaffolds for the binding and phosphorylation, and subsequent degradation of IkB. In the absence of TNFα treatment, there were more actin nodes in circular than in rectangular cells. Following TNFα treatment, the number of actin nodes increased in rectangular cells.
- Rectangular NIH3T3 cells had higher levels of MKL- a transcription regulator that binds to G-actin- in their nuclei than circular cells. When rectangular cells were treated with TNFα, MKL rapidly translocated from the nuclei to the cytoplasm.
- In rectangular NIH3T3 cells, MKL colocalized with active 5S RNA polymerase II in 50% of the imaged fractions. Colocalization reduced to as low as 1% when the SRF binding domain in MKL was deleted, indicating that SRF functions as a cofactor for the transcriptional activity of MKL. In TNFα-stimulated cells, MKL colocalization with RNA polymerase II due to the nuclear export of MKL.
- In circular NIH3T3 cells, colocalization of MKL and RNA polymerase II was very low, since most of the MKL was localized in the cytoplasm. These cells exhibited higher colocalization between p65 and RNA polymerase II.
- The expression levels of MKL target genes like alpha-smooth muscle actin (alpha-SMA) were higher in rectangular cells than in circular cells. TNFα stimulation lowered the expression of these genes in rectangular cells. The expression of NFkB target genes were higher in circular cells than in rectangular cells and TNFα stimulation further increased the expression of these genes in cells of both geometries.
- In the absence of TNFα, S-phase DNA replication was higher in circular cells than in rectangular cells. Following TNFα stimulation, rectangular cells showed significantly higher DNA replication, whereas circular cells showed no marked difference.
- Micropatterning was used to create circular or rectangular fibronectin micropatterns, on which NIH3t3 cells were seeded in order to constrain them into one of the two geometries.
- Immunostaining and confocal imaging were used to visualize the proteins under study in the NIH3T3 fibroblasts.
- Co-localization analyses were carried out on confocal images to calculate the ratios of nuclear to cytoplasmic levels of the transcription factors, p65 and NFκB.
- Real Time PCR was used to analyze gene expression patterns in NIH3T3 fibroblasts cultured on different geometrical patterns.
- The study highlights the significance of considering the intrinsic mechanical properties of a cell in determining its response to a biochemical signal.
- The study also highlights the importance of the crosstalk between mechanical and biochemical signals in maintaining cellular homeostasis within a tissue and how an aberrant crosstalk can lead to pathological states, such as fibrosis and cancer.
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