Plasticity of differentiated cells in wound repair and tumorigenesis, Part II: Skin and intestine

Recent studies have identified and begun to characterize the roles of regenerative cellular plasticity in many organs. In Part I of our two-part Review, we discussed how cells reprogram following injury to the stomach and pancreas. We introduced the concept of a conserved cellular program, much like those governing division and death, which may allow mature cells to become regenerative. This program, paligenosis, is likely necessary to help organs repair the numerous injuries they face over the lifetime of an organism; however, we also postulated that rounds of paligenosis and redifferentiation may allow long-lived cells to accumulate and store oncogenic mutations, and could thereby contribute to tumorigenesis. We have termed the model wherein differentiated cells can store mutations and then unmask them upon cell cycle re-entry the ‘cyclical hit’ model of tumorigenesis. In the present Review (Part II), we discuss these concepts, and cell plasticity as a whole, in the skin and intestine. Although differentiation and repair are arguably more thoroughly studied in skin and intestine than in stomach and pancreas, it is less clear how mature skin and intestinal cells contribute to tumorigenesis. Moreover, we conclude our Review by discussing plasticity in all four organs, and look for conserved mechanisms and concepts that might help advance our knowledge of tumor formation and advance the development of therapies for treating or preventing cancers that might be shared across multiple organs

Defining Gastric Epithelial Cell Population Dynamics At Homeostasis And Following Injury

Gastric diseases affect many people around the world, yet surprisingly little is known about the basic dynamics of gastric epithelial cells. Loss of acid-secreting parietal cells has long been observed to precede pre-cancerous gastric metaplasias like Spasmolytic Polypeptide-Expressing Metaplasia (SPEM), yet no signaling component from dying parietal cells has yet been implicated in initiating the metaplastic responses. Also, experiments pulsing 3H-thymidine and or examining intracellular components suggest that gastric mucous neck cells are short-lived transient intermediates between the gastric stem cell and mature zymogenic “chief” cells, yet specifics about this transition remain elusive. Here, we develop a novel mouse line and new techniques for tracing gastric cell lines to further probe these interactions. To identify the changes in parietal cell signaling upon injury which lead to chief cell dedifferentiation and the appearance of SPEM, we bred mice expressing the human diphtheria toxin receptor solely in parietal cells. Injection of diphtheria toxin specifically kills parietal cells through apoptosis. Surprisingly, while the parietal cells died in similar numbers to those in mice treated with tamoxifen, no SPEM or chief cell dedifferentiation was observed, and proliferation only increased through the neck, with minimal proliferation in the base. We also showed that SPEM can still arise if we inject tamoxifen or DMP-777 after the parietal cells are already killed via diphtheria toxin. These experiments indicate that chief cell dedifferentiation is not simply triggered by the loss of parietal cells, nor are dying parietal cells necessary for acute drugs to initiate metaplasia. However, the signal which initiates the metaplasia remains unknown. Furthermore, we studied the dynamics of long-lived cells in the mouse stomach using a modified BrdU pulse-chase protocol. Published reports describing gastric epithelial cell population dynamics have relied on continuous infusion or relatively short pulse-chases of DNA markers such as 3H-thymidine. Here, we pioneer a new technique, pulsing BrdU throughout our normal tamoxifen injury regimen to label nearly all cells in the unit, allowing us to chase for months and track long-lived label-retaining cells. Following our pulse of tamoxifen and BrdU, we find that nearly two thirds of chief cells retain label even through a 9-month chase, indicating that they are either longer-lived than expected or that chief cells slowly divide to maintain their own population without being replaced by newer cells from higher in the unit. We also find subpopulations of label-retaining neck cells and parietal cells exist after a 9-month chase, shedding more light on their population dynamics. To further test whether neck cells give rise to chief cells, as others have reported, we administered a short BrdU pulse followed by various chase lengths and found that most neck cells do not directly give rise to chief cells, indicating that neck cells likely have a functional, as yet unidentified purpose, other than acting as a precursor to chief cells. Finally, we show through additional tamoxifen and Helicobacter pylori injury that long-lived chief cells give rise to acute and chronic SPEM cells and find that SPEM cells can directly redifferentiate back into chief cells upon recovery from injury. Altogether, we suggest for the first time that chief cells may be a stable population in the gastric unit, largely maintaining their own census at homeostasis and in injury independently of neck cell transitions or parietal cell status

Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNA\u3csub\u3ei\u3c/sub\u3e \u3csup\u3eMet\u3c/sup\u3e Turnover in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e and Mtr4p Enzymatic Activities \u3cem\u3eIn Vitro\u3c/em\u3e

RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities

Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice

Cre/loxP technology has been widely used to study cell type-specific functions of genes. Proper interpretation of such data critically depends on a clear understanding of the tissue specificity of Cre expression. The Dmp1-Cre mouse, expressing Cre from a 14-kb DNA fragment of the mouse Dmp1 gene, has become a common tool for studying gene function in osteocytes, but the presumed cell specificity is yet to be fully established. By using the Ai9 reporter line that expresses a red fluorescent protein upon Cre recombination, we find that in 2-month-old mice, Dmp1-Cre targets not only osteocytes within the bone matrix but also osteoblasts on the bone surface and preosteoblasts at the metaphyseal chondro-osseous junction. In the bone marrow, Cre activity is evident in certain stromal cells adjacent to the blood vessels, but not in adipocytes. Outside the skeleton, Dmp1-Cre marks not only the skeletal muscle fibers, certain cells in the cerebellum and the hindbrain but also gastric and intestinal mesenchymal cells that express Pdgfra. Confirming the utility of Dmp1-Cre in the gastrointestinal mesenchyme, deletion of Bmpr1a with Dmp1-Cre causes numerous large polyps along the gastrointestinal tract, consistent with prior work involving inhibition of BMP signaling. Thus, caution needs to be exercised when using Dmp1-Cre because it targets not only the osteoblast lineage at an earlier stage than previously appreciated, but also a number of non-skeletal cell types

Mutations in Mtr4 structural domains reveal their important role in regulating tRNAi(Met) turnover in Saccharomyces cerevisiae and Mtr4p enzymatic activities in vitro

RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities

Gastric organoids: Progress and remaining challenges

The stomach is a complex and physiologically necessary organ, yet large differences in physiology between mouse and human stomachs have impeded translation of physiological discoveries and drug screens performed using murine gastric tissues. Gastric cancer (GC) is a global health threat, with a high mortality rate and limited treatment options. The heterogeneous nature of GC makes it poorly suited for current one size fits all standard treatments. In this review, we discuss the rapidly evolving field of gastric organoids, with a focus on studies expanding cultures from primary human tissues and describing the benefits of mouse organoid models. We introduce the differing methods for culturing healthy gastric tissue from adult tissues or pluripotent stem cells, discuss the promise these systems have for preclinical drug screens, and highlight applications of organoids for precision medicine. Finally, we discuss the limitations of these models and look to the future to present potential ways gastric organoids will advance treatment options for patients with GC

Plasticity of differentiated cells in wound repair and tumorigenesis, part I: stomach and pancreas

For the last century or so, the mature, differentiated cells throughout the body have been regarded as largely inert with respect to their regenerative potential, yet recent research shows that they can become progenitor-like and re-enter the cell cycle. Indeed, we recently proposed that mature cells can become regenerative via a conserved set of molecular mechanisms (‘paligenosis’), suggesting that a program for regeneration exists alongside programs for death (apoptosis) and division (mitosis). In two Reviews describing how emerging concepts of cellular plasticity are changing how the field views regeneration and tumorigenesis, we present the commonalities in the molecular and cellular features of plasticity at homeostasis and in response to injury in multiple organs. Here, in part 1, we discuss these advances in the stomach and pancreas. Understanding the extent of cell plasticity and uncovering its underlying mechanisms may help us refine important theories about the origin and progression of cancer, such as the cancer stem cell model, as well as the multi-hit model of tumorigenesis. Ultimately, we hope that the new concepts and perspectives on inherent cellular programs for regeneration and plasticity may open novel avenues for treating or preventing cancers

Mtr4p Winged Helix and Ratchet Domain Mutants Showed Defects in RNA Binding, ATP Hydrolysis and Unwinding activities.

<p>A) RNA binding of Mtr4p and Mtr4p-K904N and Mtr4p-R1030G by EMSA. Concentrations of Mtr4p used in each reaction are listed above the lanes. The arrows indicate the position(s) of Mtr4p-RNA complexes. The duplex RNA structure (R_1-4) is shown as a cartoon, indicating the migration of unbound duplex RNA, with the ³²P radiolabeled strand marked with an asterisk. B) RNA-dependent ATP hydrolysis activity of Mtr4p and Mtr4p-WH and ratchet mutants. ATPase assays were conducted with purified recombinant wild type or mutant Mtr4p at different ATP concentrations (0.25 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 8.0 mM). Near saturating levels of <i>Escherichia coli</i> total tRNA (Roche) (9 μM) was used in each reaction to initiate Mtr4p’s ATP hydrolysis activity. Rate plots of ATP hydrolysis versus ATP concentration done in triplicate are shown as best fit plots from the Michaelis-Menton equation. C) Kinetic studies to assess the efficiency of Mtr4p WH and ratchet mutants to hydrolyze ATP in the presence of near saturating levels of RNA. The K_m represents the dissociation constant of Mtr4p for ATP. k_cat/K_m provides measurement of Mtr4p ATP hydrolysis efficiency. D) Representative native-PAGE of unwinding reactions where 50 nM wild type or mutant Mtr4 protein was used in each RNA-unwinding reaction at 30°C for the times shown. Cartoons on the left indicate the migration distance of radiolabeled (*) 16 base single stranded RNA and the 22/16 duplex RNA E) Time courses of unwinding reactions for wild-type and mutant Mtr4 proteins done in triplicate are shown as plots where the proportion of single stranded RNA is plotted against reaction time and fitted to a first-order reaction. F) Shown are the kinetic parameters of RNA duplex unwinding of wild-type and WH and ratchet mutant Mtr4p, where the reaction amplitude (A) represents the fraction of unwound single-stranded RNA, and the K_obs or rate constant indicates enzyme efficiency of unwinding.</p

Structural Domain Mutant Library by Random Mutagenesis.

<p>Structural Domain Mutant Library by Random Mutagenesis.</p

The Dominant-negative Screen of Structural Domain Mutants Defective in tRNA_i^Met Turnover.

<p>A) Schematic diagram of Mtr4p showing the region of mutagenesis (double-headed arrow covering amino acid residues 610 to 1057) and the domains (<b>bold text and common pattern</b>). B) Growth test of potential dominant negative mutants. Growth was measured with a serial dilution spot assay at 33°C. The top panel shows yeast growth of <i>trm6-504</i> transformed with high-copy number plasmid YEplac195 (HC) carrying either wild type <i>MTR4</i> or no cloned DNA as negative control for suppression. The serial dilution assays shown in the bottom two panels represent a single colony picked from <i>trm6-504</i> yeast transformed with mutagenic PCR products and gapped HC plasmid. The middle panel shows examples of mutants we considered good candidates for defective Mtr4p mutants, and the bottom panel is transformants that were not considered good mutant Mtr4p candidates.</p