Activation of proHGF by St14 induces mouse embryonic stem cell differentiation
Protein Cell
Activation of proHGF by St14 induces mouse embryonic stem cell differentiation
Dear Editor
Embryonic stem cells (ESCs) are capable of unlimited selfrenewal and differentiation, thus generating remarkable interest in their potential therapeutic use in cell-based tissue engineering and regeneration. Stem cell based therapy has been considered as the most promising therapeutic strategy for a range of debilitating diseases (Reynolds and Lamba, 2014). One key requirement for stem cell differentiation and migration is to overcome existing matrix obstacles, that endowed by multiple mechanisms including tight junctions, as well as physical properties such as the matrix geometry, nanotopography, and stiffness, etc. (Guilak et al., 2009). It was reported that adherent cells showed tensile stress in the cytoskeleton by exerting contractile forces which in turn strengthen the stiffness of the matrix (Ingber, 2004). The strategy cells usually employ to disrupt the matrix obstacle is zymogen cascades mechanism that usually involved at least two proteolytic reactions that can explosively amplify the influence of extracellular signals. During this process, proteolytic activities of serine proteases, one of the most ubiquitous membrane proteases of the protease superfamily, play crucial roles by cleaving the extracellular matrix and enabling the stem cells to translocate to distant target sites (Paule et al., 2015). Facilitating the activation of extracellular signals is essential for the extracellular signaling transduction and fate decision of mESCs, as most growth factors and cytokines are usually synthesized as latent forms that require activation by the protease system located on the membrane of stem cells. Thus, the transmembrane protease system is an indispensable component of the stem cells' pluripotent program, as it plays pivotal roles in mESCs differentiation. However, the roles of transmembrane protease systems and the impact of interaction between extracellular signals and membrane protease system on pluripotency and differentiation have not been well characterized, leaving significant data gaps that hinder the therapeutic applications of stem cell therapy. Herein we explore the impact of interactions between extracellular signals and the membrane protease system through a specific transmembrane serine protease (St14), and its effect on inducing mESCs differentiation by activating proHGF, one of the substrates of St14. Once
-
cleaved at aa495, the single chain proHGF (inert form)
reverts to the active two-chain form of HGF, and mediates a
wide range of signaling pathways by binding to the c-Met
receptor
(Nishida and Hirano, 2003)
. The activation of
proHGF is important in various processes including:
placenta development, cancer progression, wound healing,
and liver regeneration
(Lee et al., 2010; Nakamura et al.,
2011)
.
To detect the expression of St14 in both pluripotent stem
cells (PSCs) and MEFs, semi-quantitative PCR and
immunoblotting were performed (Fig. 1A and 1B). Using
these approaches, we demonstrated that St14 was detected
in PSCs, but not in MEFs. This was consistent with the
results of quantitative real time PCR (96 × 96 qRT-PCR)
(Fig. 1C). Although St14 was differentially expressed in
PSCs and MEFs, their differences in the expression pattern
was not as extensive as observed in some core pluripotent
genes, such as Oct4 and Nanog.
In order to better appreciate how St14 functions in PSCs
and to examine whether it was involved in regulating
pluripotency and differentiation, the expression of select
pluripotent and differentiation genes were examined after
St14 was up-regulated in mESCs. The results demonstrated
that over-expression of St14 was correlated with decreased
expression of Oct4, both at the RNA and protein levels, while
Nanog expression was not as significantly affected
(Figs. 1D, 1E, and S1). On the other hand, over-expression
of St14 also led to increased expression of the differentiation
genes Tubb3, MyoD1, and Sox17 (Figs. 1F–H and S2). This
suggested that over-expression of St14 may be involved in
the differentiation of mESCs.
As up-regulation of St14 appeared to have an important
role in mESCs differentiation, we investigated its impact on
the activation of its substrate, proHGF, by examining mESCs
differentiation after over-expression of St14 followed by
proHGF treatment. The experimental design utilized
treatment groups that were designed as: 1) control plasmid, 2)
St14 plasmid, 3) proHGF treatment, 4) proHGF treatment +
St14 plasmid, and were used to examine the expression of
pluripotent and differentiation genes. The HGF treatment
served as the control group. The baseline level of proHGF
and HGF derived from the medium and/or endogenous
proHGF and HGF were firstly detected by an ELISA assay,
R1
iPS 1
iPS 2
iPS 3
MEF
St14
Oct4
Gapdh
St14
Oct4
Actin A B D
iPS1
iPS2
iPS3
MEF
b
in order to establish that they would not affect the
measurement of mESCs (...truncated)