Cellular Differentiation In Plants And Other Essays Of Elia

Patterns of cell growth and differentiation in cell layers can influence the quality of mature fruit. For example, pepino fruit with a compact exocarp composed of tightly packed cells are less likely to bruise during postharvest handling than cultivars having large intercellular airspaces. As cell size increases during development, other accompanying characteristics also change, such as cell wall thickness, differentiation of specific cell types (e.g. sclereids) and the formation of cell inclusions such as oil droplets or calcium oxalate crystals (raphides). In feijoa and pear, development of sclereids in the mesocarp provides the characteristic gritty texture. As another example, juiciness of orange depends on prior differentiation of juice sacs in the endocarp.

The extent and distribution of airspaces are particularly important, affecting both fruit texture and physiological properties. For instance, in apple, airspace relative to fruit volume can double during development, while cell wall thickness and relative cell surface area both decline (Figure 11.4). Such changes affect gas exchange and diffusion of solutes through pericarp tissues due to increased tortuosity.

In kiwifruit all tissues of the mature fruit (exocarp, outer and inner pericarp and central core) are already discernable in the ovary before anthesis and pollination. Each layer grows to a different extent and at different rates, so that the relative contribution of each to the total fruit volume varies with time (Figure 11.5). Cell division ceases first in the exocarp and last in the innermost regions of the central core. The outer pericarp is first seen as a homogeneous population of cells but by c. 14 d after pollination two cell types become visible, namely small isodiametric parenchyma cells full of starch grains, and much larger heavily vacuolate ovoid cells in which the frequency of starch grains per unit volume is low.

Fruit anatomy affects our perception of fruit quality. In kiwifruit, hairs are developed as multicellular projections of the skin, giving a characteristic bristly appearance and rough feel in the case of the green flesh ‘Hayward’ or a silky, smooth appearance in the yellow flesh ‘Hort16A’ cultivar. Tough skin relative to soft flesh is another important character imparted by development of primary cell wall thickenings in the hypodermal collenchyma.

1. Introduction

Stem cells are the cells that have the ability to make identical copies of themselves for the lifetime of the organism (self-renewal), and can also divide to generate progenitor or precursor cells that then differentiate into all cell types of a mature tissue (differentiation) [1]. In general, stem cells are classified into two categories depending on the plasticity of their pluripotent and differentiation potential. Embryonic stem cells (ESC) are derived from the inner cell mass of the blastocyst stage of the embryo and are pluripotent, having the ability to generate any tissue in the body but unable to generate a complete individual on their own [2,3,4]. Adult stem cells, on the other hand, are multipotent, exhibiting a more restricted differentiation capacity, and only persist in specific niches throughout postnatal life [5,6]. Certain adult stem cells such as hematopoietic, mesenchymal and neural stem cells can differentiate into multiple lineages, while others, such as endothelial and corneal stem cells are significantly lineage restricted and only possess the ability to differentiate into one cell type [7]. A delicate balance between stem cell self-renewal and differentiation defines major organ development that results in ordered layers of functional differentiated cells and residual stem cells responsible for renewal and repair [8,9].

Several studies have now confirmed the central role played by GPCRs in embryonic development and stem cell maintenance [10,11,12]. Dysregulation of these fundamental biological processes can have detrimental consequences including malignant transformation [13,14]. Increasing evidence has now demonstrated the malignant transformation of normal stem cells into cancer stem cells (CSCs) via the accumulation of various genetic modifications [15]. CSCs appear to hijack signaling pathways (e.g., GPCR) and mechanisms that regulate normal stem cells, adapting the ability to self-renew and thereby regenerating tumors after anti-cancer treatment [16,17]. While current treatment regimens kill the bulk of tumor cells, they ultimately fail to induce durable clinical responses because CSCs develop treatment resistance over time due to their quiescent nature [18,19,20,21]. Understanding the signaling pathways in normal and malignant stem cells will facilitate the use of normal stem cells for regenerative medicine and the development of new therapies to target CSCs.

2. The Diversity of GPCR Signaling Mechanisms

GPCRs are versatile signaling molecules that modulate the activities of diverse intracellular signaling via G proteins [22]. G proteins consist of Gα, Gβ and Gγ subunits, and Gα subunits have been classified into four subfamilies: Gαs, Gαi/o, Gαq/11 and Gα12/13, based on structural and functional similarities [23]. Each Gα family can relay the GPCR signals to multiple downstream effectors, consequently triggering different signaling pathways [24]. The Gαs and Gαi/o families function to activate or inhibit the activity of adenylate cyclase with a consequential increase or decrease in cyclic AMP (cAMP) production [25]. Members of the Gαq/11 family activates phospholipase-Cβ (PLCβ) ultimately leading to intracellular Ca2+ mobilization from the edoplasmic reticulum [26]. In addition, the Gα12/13 family is involved in activation of the Rho family of small GTPases [27]. These downstream effectors of G proteins subsequently trigger various intracellular signaling pathways that modulate diverse cellular functions (Figure 1) [26,28]. The major known targets of downstream G protein signaling include ion channels, calcium-sensitive enzymes, and kinases such as cAMP-dependent kinase (PKA), protein kinase X and calcium-calmodulin regulated kinases (CAMKs). Many of these kinases play contributing roles to cancer development and progression [29,30,31].

Following activation of GPCRs, rapid attenuation or desensitization of receptor responsiveness is necessary to prevent uncontrolled signaling. Desensitization is initiated by phosphorylation of the receptor by GPCR kinases [32,33] followed by uncoupling of GPCR-G protein interactions mediated by members of the β-arrestin protein family [34,35]. In addition to terminating G protein signaling, β-arrestins also play a role in promoting GPCR signaling by internalizing the receptor and acting as a molecular scaffold to recruit signaling proteins. In this way β-arrestins are capable of initiating G protein independent GPCR signaling cascades. This again challenges the traditional concept of GPCR activation involving a single ligand and receptor pair. It is now apparent that various ligands can activate a single GPCR to stabilize specific ligand-receptor conformations that promote unique signaling properties. Another critical point in the negative regulation of GPCR signaling is the deactivation of Gproteins by GTP hydrolysis, which is enhanced by Regulator of G protein Signaling Proteins (RGS). RGS proteins are capable of accelerating GTPase activity up to 1000-fold [36] and can also serve as effector agonists by competitively binding activated Gα-subunits [37,38] or promote rapid cycling of Gα-subunits between active and inactive states thereby serving as kinetic scaffolds [39]. Complex cross-talk between ligands, receptors, G proteins, second messengers, and accessory proteins facilitates the diverse range of GPCR signaling as it is recognized today.

GPCR signaling is highly diverse and the engagement of different G proteins and the strength or duration of signaling differs not only between GPCRs, but also depending on the ligand and cellular environmental context of any given GPCR. Some GPCRs, such as sphingosine-1-phosphate receptor 1 (S1P1), exclusively couple to one G protein, whereas other GPCRs, such as lysophosphatidic acid receptors (LPA), can couple to multiple G proteins triggering diverse downstream signaling cascades [40,41]. Aberrant activation of GPCR signaling triggered by high-affinity ligands (e.g., LPA and S1P1) leads to malignant transformation, proliferation, metastasis and drug resistance [42,43].

2.1. GPCR-Mediated Regulation of Stem Cell Properties

The role of GPCR signaling in stem cell function, although undoubtedly important, has not been fully elucidated. Several lines of evidence are suggestive of a critical role, for instance many of the signaling cascades activated by GPCR signaling directly regulate, or are synergistic with, pathways that regulate ESC pluripotency and differentiation [49]. General roles for G proteins in regulating pluripotency have also been described. Gαs signaling has been shown to promote proliferation and pluripotency in self-renewing and differentiating mouse ESCs [11]. Signaling mediated by Gαi proteins is demonstrated to affect the morphology and organization of human induced pluripotent stem cells (iPSC) [10]. Dramatic changes in the expression levels of GPCRs in distinct stages of stem cell differentiation further implicate their involvement in stem cell function [50]. Comprehensive qPCR analysis of more than 350 GPCR genes between three stages of in vitro neural differentiation (i.e., pluripotent human ESCs, multipotent neural progenitors, and differentiated neurons) has revealed striking differences in GPCR expression within the different cell populations [50,51].

The GPCR superfamily is divided into five sub-families, including glutamate, frizzled, adhesion, rhodopsin and secretin. The specific roles of GPCRs from two of these families (i.e., frizzled and rhodospin) will be discussed in this review exemplifying GPCR-mediated regulation of normal and malignant stem cells.

2.2. Wnt-Activated Fzd Signaling

The frizzled (Fzd) family of GPCRs is activated by the Wnt family of lipoglycoproteins [52]. Wnt signaling plays a critical role during development and Wnt ligands are known to regulate the maintenance of numerous stem cell populations in both developing and adult organisms [53]. Although Fzds have normal GPCR topology, their lack of sequence similarity leads to debate regarding their classification as GPCRs [54]. Nevertheless, compelling experimental observations show that heterotrimeric G proteins play a crucial role in Wnt signaling, specifically given that Wnt signaling activation could be abrogated in human ESCs by a Gαi/o protein inhibitor (pertussis toxin) [55,56]. Furthermore, Gαo has been shown to be essential for Wnt activation in Drosophila [57]. As such, the Fzd family is listed by the International Union of Pharmacology as a novel and separate family of GPCRs termed “Class Frizzled” [58].

Nineteen Wnt proteins serve as the primary endogenous agonists for 10 Fzd receptors encoded in the human genome [59]. There is apparent specificity between individual Fzds and their ligands with Wnt3a-Fzd1, Wnt5a-Fzd7 and Wnt7-Fzd6 being identified as highly efficient Wnt-Fzd pairs [60,61]. Three main pathways in Wnt-activated Fzd signaling include: Fzd/Ca2+ pathway, Fzd/planar cell polarity (PCP) pathway and Fzd/β-catenin pathway. Agonist stimulation of the Fzd/Ca2+ pathway leads to elevated intracellular Ca2+ levels in a G protein-dependent manner that activates calcium-dependent protein kinase c (PKC) and Ca2+/calmodulin-dependent protein kinase [62,63]. The Fzd/PCP pathway tranduces via Dishevelled (Dvl) to small Rho GTPases and their effectors Rho-associated coiled-coil containing protein kinase (ROCK) and the c-Jun-N-terminal kinase/c-Jun/AP-1 pathway [64]. Agonist stimulation in the Fzd/β-catenin pathway activates the phosphoprotein Dvl, leading to inhibition of the destruction complex composed of adenomatosis polyposis coli protein (APC) and Axin. β-catenin then translocates from the cytoplasm to the nucleus, where it cooperates with the T-cell factor/lymphoid enhancer factor (Tcf/Lef) transcription factors to modify transcription of a set of Wnt target genes [53]. The Wnt/Fzd pathways have been classified as regulators of cell fate determination and control cell movement and tissue polarity, respectively [49].

Fzd receptors play an important role in mammalian development and stem cell self-renewal. The expression of Fzd5, 7 and 10 has been found in the gastrulating embryos of mice and is implicated in neural induction [65]. Evidence from knockout mouse studies suggests that Fzd4, 5 and 9 are important for central nervous system development and self-renewal of B cell populations [66,67,68]. Various studies suggest that Wnt3a inhibitor or GSK-3 inhibitor (6-bromoindirubin-3’-oxime) maintains pluripotency in human ESCs [69]. In particular, the mRNA levels of the Wnt receptor Fzd7 are found to be 200-fold higher in human ESCs compared to differentiated cell types, and Fzd7 knockdown induces significant morphological changes in ESC colonies with concomitant loss of the pluripotency gene octamer-binding protein 4 (Oct-4) [70]. In contrast, some studies have reported a pro-differentiation role for Wnt/Fzd signaling [71,72,73] suggesting a cellular context-dependent effect of Wnt signaling on stem cell self-renewal.

Wnt/β-catenin signaling is evolutionarily conserved and plays critical roles in development and disease [74]. Inappropriate pathway activation produces uncontrolled cell growth leading to cancer, and aberrant Wnt signaling has been implicated in different types of cancer, including hepatcellular carcinomas, ovarian carcinomas, leukemia, prostate cancers, colon cancers and melanoma [75]. Indeed, the extent to which carcinomas rely on Wnt signaling to drive their development and progression is exemplified by the observation that approximately 90% of colon cancer and 50% of breast cancer cases are associated with hyperactivation of Wnt signaling [76,77].

Dysregulation of Wnt/β-catenin signaling occurs in multiple types of CSCs and increasing evidence has demonstrated a crucial role for Wnt/β-catenin signaling in the self-renewal and malignant behavior of CSCs [78,79,80,81]. We as well as others have previously demonstrated that aberrant activation of Wnt/β-catenin signaling contributes to the transformation of hematopoietic stem cells (HSCs) into leukemic stem cells (LSCs) [78,82]. Furthermore, our laboratory has recently identified GPR84 as a novel regulator of β-catenin signaling in LSCs [83]. GPR84 overexpression induces the activation of β-catenin transcriptional co-factors Tcf7l2 and c-Fos, as well as a gene set associated with Wnt signaling [69]. Our functional study shows that GPR84 depletion impairs LSC function and inhibits the development of an aggressive and drug-resistant subtype of acute myeloid leukemia (AML) [83]. Importantly, the GPR84-deficient phenotype is β-catenin dependent as re-expression of active β-catenin is capable of rescuing the deficiency [69]. In addition, levels of GPR84 expression are significantly upregulated in human and mouse AML LSCs compared to normal HSCs [83], providing a therapeutic window to selectively target LSCs while sparing normal HSCs. These studies demonstrate a strong rationale for inhibiting GPCR/β-catenin signaling as a novel therapeutic strategy to target drug-resistant malignant stem cells in cancer.

In support of this therapeutic rationale, we have recently shown an essential role for G protein subunit Gαq in the maintenance of AML LSCs [84]. By using both shRNA-mediated silencing and pharmacological inhibition, our study shows that Gαq regulates LSC growth and survival in vitro and in vivo, and controls β-catenin activity. Using a commercially available Gαq inhibitor, GP-antagonist 2A, our data indicates that ex vivo pre-treatment of LSCs with the antagonist impairs their proliferative capacity in mouse bone marrow and prolongs mouse survival [84]. Therefore, further investigations into the therapeutic applicability of GP-antagonist 2A for the treatment of AML are significantly warranted. In addition, we have shown that inhibiting Gαq expression leads to suppression of mitochondrial complex 1 subunits (i.e., Nd2, Nd4l, Nd5) and consequent disruptions in mitochondrial function and energy metabolism in leukemic cells [84], providing a mechanism linking mitochondrial dysfunction with leukemogenesis via Gαq signaling activation. Thus, targeting β-catenin signaling and energy metabolism by blocking Gαq signaling could represent a novel therapeutic approach to reduce leukemogenesis in aggressive AML.

2.3. Rhodospin Class of GPCRs

The rhodopsin class is by far the largest GPCR family, comprising almost 85% of GPCRs. The leucine-rich repeat-containing (Lgr) proteins are a distinct subset of evolutionarily conserved rhodopsin GPCRs containing a large extracellular domain with multiple leucine-rich repeats [85]. The Lgr family member Lgr5 is a known stem cell marker in certain types of tissue [86,87]. In vivo lineage tracing experiments using a heritable-inducible lacZ reporter gene introduced into Lgr5-expressing cells has shown that Lgr5 is a marker of adult intestinal stem cells. Further examination of Lgr5 expression patterns in mice has identified discrete populations of Lgr5-expressing cells in organs including skin, stomach, mammary gland, tongue, kidney and endometrium, indicating that Lgr5 may function as a universal epithelial stem cell marker [86,88,89,90,91].

Epithelial homeostasis in the adult intestine is regulated by several signaling pathways and key among these is the Wnt signaling pathway [92]. Hyperactivation of the Wnt signaling pathway is associated with transformation of the intestinal epithelium [93]. Lgr5 has been identified as a Wnt target gene and overexpression of Lgr5 antagonizes Wnt signaling [94,95,96]. The exact mechanism remains unknown but the potential outcome of Lgr5 antagonism would result in β-catenin phosphorylation and targeting for degradation [76]. In addition, overexpression of Lgr5 in colon cancer and HEK293 cells decreases cell motility and stimulates cell-cell adhesion [97]. R-spondin proteins (Rspo1-4) have been identified as ligands of the Lgr family [98]. The inhibitory effect of Lgr5 appears to be abolished in the presence of Rspo [76], and one potential model for potentiation of Wnt involves direct interaction and formation of a Wnt-potentiating complex, Rspo/Lgr5/Wnt/Fzd, at the plasma membrane [94]. Two highly homologous Wnt target genes, Rnf43 and Znrf3, also play a role in the complex regulation of Wnt signaling at the receptor level. Both Rnf43 and Znrf3 are ubiquitin ligases found specifically in Lgr5 crypt stem cells and enriched in colon cancer [99,100,101]. These ubiquitin ligases mediate multiubiquitination of lysines in the cytoplasmic transmembrane domains of Fzds that results in rapid endocytosis of Wnt receptors and their destruction by lysosomes. Loss of Rnf43 and Znrf3 expression results in hyperresponsiveness to Wnt signals leading to the formation of abnormal adenomas consisting entirely of Lgr5 stem cells [100]. Since Rnf43 and Znrf3 are encoded by Wnt target genes, this represents an intricate negative feedback loop controlling Wnt receptor expression [81]. Furthermore, it has been demonstrated that the Rnf43/Znrf3-mediated membrane clearance of Wnt receptors can be reversed by R-spondin [101], and thus Rspo-Lgr complexes neutralize Rnf43/Znrf3 to allow persistence of Fzds receptors and boosting Wnt signal strength (Figure 2).

A recent study by Baker et al. has demonstrated the role of Lgr5-expressing cells in the development of colon cancer. By applying in situ hybridization (ISH) to a panel of human normal colon, adenoma and carcinoma samples, significant increase in levels of Lgr5 mRNA is observed in all serrated lesions that is accompanied by expansion of proliferative and invasive compartments, suggesting that Lgr5 may support invasion and metastasis [102]. Within the colonic crypts, many cells are endowed with stem cell potential but only a small percentage of the total Lgr5-expressing cells actually functions as stem cells at any one time [103]. Within the malignant adenomatous crypts, however, dysregulation of the processes that govern normal stem cell maintenance results in an elevated number of functional stem cells [104]. Deducing the connection between Wnt signaling, Lgr5 signaling and cancer cell migration will improve our understanding of the role for GPCR signaling in migration of normal and malignant stem cells.

Interestingly, Lgr4 and Lgr6, two close family members of Lgr5, have also been implicated in the regulation of stem cell properties. Lgr6 expression is found to mark hair follicle epidermal stem cells, whereas Lgr4 expression plays a major role in prostate stem cell function [79,105

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