Hemopoietic regulators.

The production and maturation of blood cells from the eight major blood cell lineages is a complex and continuous process, which is largely controlled by specific glycoprotein hemopoietic regulators. These regulators also control the functional activity of the blood cells through eliciting a diverse set of intracellular responses initiated by a regulator-specific membrane receptor. Twenty of these regulators have now been characterized, and their mass production has led to four already being licensed for clinical use in disease states involving subnormal blood cell formation.     

Genetic and epigenetic regulators of pluripotency

Genetic and epigenetic mechanisms regulate the transition from the totipotent zygote to pluripotent primitive ectoderm cells in the inner cell mass of mouse blastocysts. These pluripotent cells can be propagated indefinitely in vitro, underpinned by a unique epigenetic state. Following implantation of the blastocyst, diverse epigenetic modifiers control differentiation of pluripotent epiblast cells into somatic cells, while specification of germ cells requires repression of the somatic program. Regenerating totipotency during development of germ cells entails re-expression of pluripotency-specific genes and extensive erasure of epigenetic modifications. Increasing knowledge of key underlying mechanisms heightens prospects for creating pluripotent cells directly from adult somatic cells.     

Podosomes as smart regulators of cellular adhesion

Podosomes are punctate adhesion structures first described in osteoclasts and next found in src-transformed cells of mesenchymal origin. Podosomes were never observed in cultured epithelial cells where cell-matrix adhesion structures were represented only by focal contacts and hemidesmosomes interacting with microfilaments and intermediate filaments, respectively. Rat bladder carcinoma cells and normal human keratinocytes showed that hemidesmosome-like structures are organized around a core of actin filaments that appears early during cell adhesion and looks similar to those of podosomes described in cells of mesenchymal origin. The epithelial podosome-like structures specifically contain Arp2/3 complex, cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin and zyxin. The maintenance of the F-actin core and the surrounding hemidesmosomes depends on actin polymerization, src family kinases and Grb2, but not on microtubular integrity. Thus, podosomes are not unique to cells of mesenchymal origin, but also appear in epithelial cells where they may take part in regulating basement membrane adhesion.     

Apoptosis regulators

Over the last decade, a great deal of attention has been directed at elucidating the role of apoptosis regulators in governing survival decisions in neoplastic cells, particularly those of hematopoietic origin. A major focus of this work has involved investigation of the function of pro- and anti-apoptotic members of the BCL-2 family, and the relationship between these proteins and mitochondrial integrity. Currently, these proteins can be classified into two broad categories: those that modulate mitochondrial function and those that regulate the activation of caspases responsible for activation and execution of the apoptotic cascade. Within the first category, certain proteins (e.g., BCL-2, BCL-xL) act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or release of pro-apoptotic proteins such as cytochrome C into the cytosol. Other proapoptotic proteins (e.g., BAX, BAK, BIM) promote release of cytochrome C. These proteins are therefore primarily involved in regulation of the intrinsic, mitochondrial apoptotic pathway. Within the second category, proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP block the activation of caspases, particularly those involved in engagement of the receptor-related, extrinsic apoptotic pathway. Cross-talk between the intrinsic and extrinsic pathways exists. For example, the BH3-domain only protein BID is cleaved by the activation of pro-caspase-8 through the extrinsic pathway, and translocates to the mitochondrion to promote cytochrome C release. Apoptosis is also regulated by various signal transduction pathways, possibly through post-translational modifications in BCL-2 family proteins. For example, phosphorylation of BCL-2 through a JNK-dependent mechanism has been postulated to contribute to apoptosis induced by the taxane class of cytotoxic agents. Finally, attempts to modulate apoptotic pathways with small molecules have recently received much attention. For example, small molecule inhibitors of BCL-2 or mimetics of SMAC/DIABLO, which opposes the actions of XIAP, have recently been shown to promote the antineoplastic activity of conventional cytotoxic agents. It is likely that an improved understanding of apoptosis regulation will lead to new insights into neoplastic transformation, and may also provide important leads for the development of novel antileukemic strategies.     

Peptide regulators of peripheral taste function

The peripheral sensory organ of the gustatory system, the taste bud, contains a heterogeneous collection of sensory cells. These taste cells can differ in the stimuli to which they respond and the receptors and other signaling molecules they employ to transduce and encode those stimuli. This molecular diversity extends to the expression of a varied repertoire of bioactive peptides that appear to play important functional roles in signaling taste information between the taste cells and afferent sensory nerves and/or in processing sensory signals within the taste bud itself. Here, we review studies that examine the expression of bioactive peptides in the taste bud and the impact of those peptides on taste functions. Many of these peptides produced in taste buds are known to affect appetite, satiety or metabolism through their actions in the brain, pancreas and other organs, suggesting a functional link between the gustatory system and the neural and endocrine systems that regulate feeding and nutrient utilization.     

Flavonoids as developmental regulators

Flavonoids, usually regarded as dispensable phytochemicals derived from plant secondary metabolism, play important roles in the biology of plants by affecting several developmental processes. Bioactive flavonoids also signal to microbes, serve as allelochemicals and are important nutraceuticals in the animal diet. Despite the significant progress made in identifying flavonoid pathway genes and regulators, little is currently known about the protein targets of flavonoids in plant or animal cells. Recently, there have been advances in our understanding of the roles that flavonoids play in developmental processes of plants. The multiple cellular roles of flavonoids can reflect their chemical diversity, or might suggest the existence of cellular targets shared between many of these seemingly disparate processes.     

Negative regulators of growth.

The proliferation of cells is regulated by countervailing positively- and negatively-acting signaling networks. The anti-proliferative signals, the study of which has been much neglected until recently, are often conveyed by growth-inhibitory peptides. Elements that mediate the cellular response to growth inhibitors are encoded by tumor suppressor genes that if lost may lead to the runaway growth of the cancer cell.     

Thrombospondins function as regulators of angiogenesis

Thrombospondins (TSPs) -1 and -2 were among the first protein inhibitors of angiogenesis to be identified, a property that was subsequently attributed to the interactions of sequences in their type I repeats with endothelial cell-surface receptors. The interactions of TSPs-1 and -2 with cell-surface receptors, proteases, growth factors, and other bioactive molecules, coupled with the absence of direct structural functions that can be attributed to these matrix proteins, qualify them for inclusion in the category of ‘matricellular proteins’. The phenotypes of TSP-1, TSP-2, and double TSP-1/2-null mice confirm the roles that these proteins play in the regulation of angiogenesis, and provide clues to some of the other important functions of these multi-domain proteins. One of these functions is the ability of TSP-1 to activate the latent TGFβ1 complex, a property that is not shared by TSP-2. A major pathway by which TSP1 or TSP2 inhibits angiogenesis involves an interaction with CD 36 on endothelial cells, which leads to apoptosis of both the liganded and adjacent cells. However a homeostatic mechanism, which inhibits endothelial cell proliferation, and may be physiologically preferable under some circumstances, has also been elucidated, and involves interaction with the very low density lipoprotein receptor (VLDLR). The interaction of TSP1with its receptor, CD47, further inhibits angiogenesis by antagonizing nitric oxide signaling in endothelial and vascular smooth muscle cells. Paradoxically, there is also evidence that TSP-1 can function to promote angiogenesis. This apparent contradiction can be explained by the presence of sequences in different domains of the protein that interact with different receptors on endothelial cells. The anti-angiogenic function of TSPs has spurred interest in their use as anti-tumor agents. Currently, peptide mimetics, based on sequences in the type I repeats of TSPs that have been shown to have anti-angiogenic properties, are undergoing clinical testing.     

Negative regulators of programed cell death.

During animal development many cells undergo programed deaths. Recently, genes that suppress the cell-death program have been described in both vertebrates and invertebrates. These genes play a vital role in regulation of the molecules that kill cells, and disruption of this regulatory process can contribute to disease.     

Key regulators in neuronal polarity

Neurons are highly polarized cells, most of which develop a single axon and several dendrites. These two compartments acquire specific characteristics that enable neurons to transmit intercellular signals from several dendrites to an axon. A wealth of recent studies has shown that PI 3-kinase, Rho family GTPases, the Par complex, and cytoskeleton-related proteins participate in the initial events of neuronal polarization. Here, we review the role of polarity-regulating molecules and the potential mechanisms underlying the specification of an axon and dendrites.     

Schwann cells as regulators of nerve development.

Myelinating and non-myelinating Schwann cells of peripheral nerves are derived from the neural crest via an intermediate cell type, the Schwann cell precursor [K.R. Jessen, A. Brennan, L. Morgan, R. Mirsky, A. Kent, Y. Hashimoto, J. Gavrilovic. The Schwann cell precursor and its fate: a study of cell death and differentiation during gliogenesis in rat embryonic nerves, Neuron 12 (1994) 509-527]. The survival and maturation of Schwann cell precursors is controlled by a neuronally derived signal, beta neuregulin. Other factors, in particular endothelins, regulate the timing of precursor maturation and Schwann cell generation. In turn, signals derived from Schwann cell precursors or Schwann cells regulate neuronal numbers during development, and axonal calibre, distribution of ion channels and neurofilament phosphorylation in myelinated axons. Unlike Schwann cell precursors, Schwann cells in older nerves survive in the absence of axons, indicating that a significant change in survival regulation occurs. This is due primarily to the presence of autocrine growth factor loops in Schwann cells, present from embryo day 18 onwards, that are not functional in Schwann cell precursors. The most important components of the autocrine loop are insulin-like growth factors, platelet derived growth factor-BB and neurotrophin 3, which together with laminin support long-term Schwann cell survival. The paracrine dependence of precursors on axons for survival provides a mechanism for matching precursor cell number to axons in embryonic nerves, while the ability of Schwann cells to survive in the absence of axons is an absolute prerequisite for nerve repair following injury. In addition to providing survival factors to neurones and themselves, and signals that determine axonal architecture, Schwann cells also control the formation of peripheral nerve sheaths. This involves Schwann cell-derived Desert Hedgehog, which directs the transition of mesenchymal cells to form the epithelium-like structure of the perineurium. Schwann cells thus signal not only to themselves but also to the other cellular components within the nerve to act as major regulators of nerve development.     

Tropomodulins are negative regulators of neurite outgrowth

Regulation of the actin cytoskeleton is critical for neurite formation. Tropomodulins (Tmods) regulate polymerization at actin filament pointed ends. Previous experiments using a mouse model deficient for the neuron specific isoform Tmod2 suggested a role for Tmods in neuronal function by impacting processes underlying learning and memory. However, the role of Tmods in neuronal function on the cellular level remains unknown. Immunofluorescence localization of the neuronal isoforms Tmod1 and Tmod2 in cultured rat primary hippocampal neurons revealed that Tmod1 is enriched along the proximal part of F-actin bundles in lamellipodia of spreading cells and in growth cones of extending neurites, while Tmod2 appears largely cytoplasmic. Functional analysis of these Tmod isoforms in a mouse neuroblastoma N2a cell line showed that knockdown of Tmod2 resulted in a significant increase in the number of neurite-forming cells and in neurite length. While N2a cells compensated for Tmod2 knockdown by increasing Tmod1 levels, over-expression of exogenous Tmod1 had no effect on neurite outgrowth. Moreover, knockdown of Tmod1 increased the number of neurites formed per cell, without effect on the number of neurite-forming cells or neurite length. Taken together, these results indicate that Tmod1 and Tmod2 have mechanistically distinct inhibitory roles in neurite formation, likely mediated via different effects on F-actin dynamics and via differential localizations during early neuritogenesis.     

Glycosaminoglycans as regulators of stem cell differentiation

ES (embryonic stem) cell differentiation is dependent on the presence of HS (heparan sulfate). We have demonstrated that, during differentiation, the evolution of specific cell lineages is associated with particular patterns of GAG (glycosaminoglycan) expression. For example, different HS epitopes are synthesized during neural or mesodermal lineage formation. Cell lines mutant for various components of the HS biosynthetic pathway are selectively impaired in their differentiation, with lineage-specific effects observed for some lines. We have also observed that the addition of soluble GAG saccharides to cells, with or without cell-surface HS, can influence the pace and outcome of differentiation, again highlighting specific pattern requirements for particular lineages. We are combining this work with ongoing studies into the design of artificial cell environments where we have optimized three-dimensional scaffolds, generated by electrospinning or by the formation of hydrogels, for the culture of ES cells. By permeating these scaffolds with defined GAG oligosaccharides, we intend to control the mechanical environment of the cells (via the scaffold architecture) as well as their biological signalling environment (using the oligosaccharides). We predict that this will allow us to control ES cell pluripotency and differentiation in a three-dimensional setting, allowing the generation of differentiated cell types for use in drug discovery/testing or in therapeutics.     

Transglutaminases: key regulators of cancer metastasis

The ability to metastasize represents the most important characteristic of malignant tumors. The biological details of the metastatic process remain somewhat unknown, due to difficulties in studying tumor cell behaviour with high spatial and temporal resolution in vivo. Several lines of evidence involve transglutaminases (TGs) in the key stages of tumor progression cascade, even though the molecular mechanisms remain controversial. TG expression and activity display a different role in the primary tumor or in metastatic cells. In fact, TG expression is low in the primary tumor mass, but augmented when cells acquire the metastatic phenotype. Nevertheless, in other cases, the use of inducers of TG transamidating activity seems to contrast tumor cell plasticity, migration and invasion. In the following review, the function of TGs in cancer cell migration into the extracellular matrix, adhesion to the capillary endothelium and its basement membrane, invasion and angiogenesis is discussed.