Vimentin intermediate filaments modulate cell traction force but not cell sensitivity to substrate stiffness.

The ability of cells to sense and respond to the mechanical stiffness of the surrounding matrix is important to support normal cell function, wound healing, and development. Central to the process of durosensing is the cytoskeleton composed of three classes of filaments: F-actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that contributes significantly to cell mechanics and cell traction force, which is required to probe extracellular matrix. The role of vimentin in how cells sense and respond to the mechanical rigidity of extracellular matrix is largely unclear. To investigate the role of vimentin in durosensing, we knocked down the vimentin expression level in 3T3 fibroblasts using shRNA transfection and measured cellular responses as functions of substrate stiffness. We quantified durosensitivity by the rates at which cell area and traction force change with substrate stiffness. Our results show that that vimentin plays a role in durosensing by modulating traction force and knocking out vimentin did not significantly affect durosensitivity. These results indicate that vimentin may be a redundant component of the machinery that cells use to sense substrate stiffness.

High molecular weight FGF2 isoforms demonstrate canonical receptor-mediated activity and support human embryonic stem cell self-renewal.

Basic fibroblast growth factor (FGF2) is a highly pleiotropic member of a large family of growth factors with a broad range of activities, including mitogenesis and angiogenesis (Ornitz et al., 1996; Zhang et al., 2006), and it is known to be essential for maintenance of balance between survival, proliferation, and self-renewal in human pluripotent stem cells (Eiselleova et al., 2009; Zoumaro-Djayoon et al., 2011). A single FGF2 transcript can be translated into five FGF2 protein isoforms, an 18kDa low molecular weight (LMW) isoform and four larger high molecular weight (HMW) isoforms (Arese et al., 1999; Arnaud et al., 1999). As they are not generally secreted, high molecular weight (HMW) FGF2 isoforms have predominantly been investigated intracellularly; only a very limited number of studies have investigated their activity as extracellular factors. Here we report over-expression, isolation, and biological activity of all recombinant human FGF2 isoforms. We show that HMW FGF2 isoforms can support self-renewal of human embryonic stem cells (hESCs) in vitro. Exogenous supplementation with HMW FGF2 isoforms also activates the canonical FGFR/MAPK pathway and induces mitogenic activity in a manner similar to that of the 18kDa FGF2 isoform. Though all HMW isoforms, when supplemented exogenously, are able to recapitulate LMW FGF2 activity to some degree, it appears that certain isoforms tend to do so more poorly, demonstrating a lesser functional response by several measures. A better understanding of isoform-specific FGF2 effects will lead to a better understanding of developmental and pathological FGF2 signaling.

FGF2 Overrides TGFß1-Driven Integrin ITGA11 Expression in Human Dermal Fibroblasts.

Deposition of collagen-based extracellular matrix by fibroblasts during wound healing leads to scar formation--a typical outcome of the healing process in soft tissue wounds. The process can, however, be skewed in favor of tissue regeneration by manipulation of wound environment. Low oxygen conditions and supplementation with FGF2 provide extracellular cues that drive wound fibroblasts towards a pro-regenerative phenotype. Under these conditions, fibroblasts dramatically alter expression of many genes among which the most significantly deregulated are extracellular matrix and adhesion molecules. Here we investigate the mechanism of a collagen I binding integrin α11 (ITGA11) deregulation in response to low oxygen-mediated FGF2 effects in dermal fibroblasts. Using RT-PCR, qRT-PCR, Western blotting, and immunocytochemistry, we describe significant down-regulation of ITGA11. Decrease in ITGA11 is associated with its loss from focal adhesions. We show that loss of ITGA11 requires FGF2 induced ERK1/2 activity and in the presence of FGF2, ITGA11 expression cannot be rescued by TGFß1, a potent activator of ITGA11. Our results indicate that FGF2 may be redirecting fibroblasts towards an anti-fibrotic phenotype by overriding TGFß1 mediated ITGA11 expression.

Expression and differentiation between OCT4A and its Pseudogenes in human ESCs and differentiated adult somatic cells.

The POU5F1 gene codes for the OCT4 transcription factor, which is one of the key regulators of pluripotency. Its transcription, alternative splicing, and alternative translation leading to the synthesis of the active, nuclear localized OCT4A has been described in detail. Much less, however, is known about actively transcribed OCT4 pseudogenes, several of which display high homology to OCT4A and can be expressed and translated into proteins. Using RT-PCR followed by pseudogene-specific restriction digestion, cloning, and sequencing we discriminate between OCT4A and transcripts for pseudogenes 1, 3 and 4. We show that expression of OCT4 and its pseudogenes follows a developmentally-regulated pattern in differentiating hESCs, indicating a tight regulatory relationship between them. We further demonstrate that differentiated human cells from a variety of tissues express exclusively pseudogenes. Expression of OCT4A can, however be triggered in adult differentiated cells by oxygen and FGF2-dependent mechanisms.