Mutations in SALL4, the human homolog of the Drosophila homeotic gene spalt (sal), cause the autosomal dominant disorder known as Okihiro syndrome. In this study, we show that a targeted null mutation in the mouse Sall4 gene leads to lethality during peri-implantation. Growth of the inner cell mass from the knockout blastocysts was reduced, and Sall4-null embryonic stem (ES) cells proliferated poorly with no aberrant differentiation. Furthermore, we demonstrated that anorectal and heart anomalies in Okihiro syndrome are caused by Sall4 haploinsufficiency and that Sall4/Sall1 heterozygotes exhibited an increased incidence of anorectal and heart anomalies, exencephaly and kidney agenesis. Sall4 and Sall1 formed heterodimers, and a truncated Sall1 caused mislocalization of Sall4 in the heterochromatin; thus, some symptoms of Townes-Brocks syndrome caused by SALL1 truncations could result from SALL4 inhibition.
Ectodomain shedding mediated by tumor necrosis factor–α (TNF-α)–converting enzyme [TACE; also known as ADAM17 (a disintegrin and metalloproteinase 17)] provides an important switch in regulating cell proliferation, inflammation, and cancer progression. TACE-mediated ectodomain cleavage is activated by signaling of the mitogen-activated protein kinases (MAPKs) p38 and ERK (extracellular signal– regulated kinase). Here, we found that under basal conditions, TACE was predominantly present as dimers at the cell surface, which required its cytoplasmic domain and enabled efficient association with tissue inhibitor of metalloproteinase-3 (TIMP3) and silencing of TACE activity. Upon activation of the ERK or p38 MAPK pathway, the balance shifted from TACE dimers to monomers, and this shift was associated with increased cell surface presentation of TACE and decreased TIMP3 association, which relieved the inhibition of TACE by TIMP3 and increased TACE-mediated proteolysis of transforming growth factor–α. Thus, cell signaling altered the dimer-monomer equilibrium and inhibitor association to promote activation of TACE-mediated ectodomain shedding, a regulatory mechanism that may extend to other ADAM proteases.
Background The diversity of cell types and tissue types that originate throughout development derives from the differentiation potential of embryonic stem cells and somatic stem cells. While the former are pluripotent, and thus can give rise to a full differentiation spectrum, the latter have limited differentiation potential but drive tissue remodeling. Additionally cancer tissues also have a small population of self-renewing cells with stem cell properties. These cancer stem cells may arise through dedifferentiation from non-stem cells in cancer tissues, illustrating their plasticity, and may greatly contribute to the resistance of cancers to chemotherapies. Scope of review The capacity of the different types of stem cells for self-renewal, the establishment and maintenance of their differentiation potential, and the selection of differentiation programs are greatly defined by the interplay of signaling molecules provided by both the stem cells themselves, and their microenvironment, the niche. Here we discuss common and divergent roles of TGF-β family signaling in the regulation of embryonic, reprogrammed pluripotent, somatic, and cancer stem cells. Major conclusions Increasing evidence highlights the similarities between responses of normal and cancer stem cells to signaling molecules, provided or activated by their microenvironment. While TGF-β family signaling regulates stemness of normal and cancer stem cells, its effects are diverse and depend on the cell types and physiological state of the cells. General significance Further mechanistic studies will provide a better understanding of the roles of TGF-β family signaling in the regulation of stem cells. These basic studies may lead to the development of a new therapeutic or prognostic strategies for the treatment of cancers. This article is part of a Special Issue entitled Biochemistry of Stem Cells.
Background: TGF- signaling is required for primed pluripotency, but the roles of Smad2 and Smad3 have not been well defined. Results: Smad2, but not Smad3, has a role in pluripotency by activating Nanog expression and repressing BMP signaling. Conclusion: Smad2 is essential in the maintenance of pluripotency. Significance: The roles of Smad2 and Smad3 need to be distinguished in the regulation of pluripotency by TGF- signaling.
Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling.
Mesenchymal stromal cells (MSCs) have been isolated from numerous sources and are potentially therapeutic against various diseases. Umbilical cord‐derived MSCs (UC‐MSCs) are considered superior to other tissue‐derived MSCs since they have a higher proliferation rate and can be procured using less invasive surgical procedures. However, it has been recently reported that 2D culture systems, using conventional cell culture flasks, limit the mass production of MSCs for cell therapy. Therefore, the development of alternative technologies, including microcarrier‐based cell culture in bioreactors, is required for the large‐scale production and industrialization of MSC therapy. In this study, we aimed to optimize the culture conditions for UC‐MSCs by using a good manufacturing practice (GMP)‐compatible serum‐free medium, developed in‐house, and a small‐scale (30 mL) bioreactor, which was later scaled up to 500 mL. UC‐MSCs cultured in microcarrier‐based bioreactors (MC‐UC‐MSCs) showed characteristics equivalent to those cultured statically in conventional cell culture flasks (ST‐UC‐MSCs), fulfilling the minimum International Society for Cellular Therapy criteria for MSCs. Additionally, we report, for the first time, the equivalent therapeutic effect of MC‐UC‐MSCs and ST‐UC‐MSCs in immunodeficient mice (graft‐versus‐host disease model). Lastly, we developed a semi‐automated cell dispensing system, without bag‐to‐bag variation in the filled volume or cell concentration. In summary, our results show that the combination of our GMP‐compatible serum‐free and microcarrier‐based culture systems is suitable for the mass production of MSCs at an industrial scale. Further improvements in this microcarrier‐based cell culture system can contribute to lowering the cost of therapy and satisfying several unmet medical needs.
Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are inflammatory eczematous skin diseases caused by various factors. Here, we report that topical application of the dipeptide, L-glutamic acid-L-tryptophan (L-Glu-L-Trp), improved symptoms in both ACD and AD in mice. Using a mouse model of ACD induced by repeated application of 2,4-dinitorofluorbenzene (DNFB), we demonstrated that L-Glu-L-Trp attenuated DNFB-induced skin thickening. In addition, quantification of cytokines in serum revealed that L-Glu-L-Trp suppressed the DNFBinduced increase in the interleukin (IL)-22 level. Moreover, L-Glu-L-Trp attenuated mite antigen extractinduced AD model symptoms such as the increase of skin thickening and elevation of serum IL-22. We also confirmed that the dipeptide structure rather than the individual amino acid components was important for the therapeutic effects of L-Glu-L-Trp. Furthermore, we showed that IL-22 decreased the expression level of filaggrin mRNA in human epidermal keratinocytes, and L-Glu-L-Trp attenuated that effect. These results suggested that the topical application of the dipeptide, L-Glu-L-Trp, to the skin may be useful for treating ACD and AD.
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