Under what condition will a target cell respond quickly to an extracellular signal?

Other Signal Transduction and Cell Cycle Regulation Pathways

Aberrant activation of additional signal transduction pathways in RCC may also contribute to altered cell cycle kinetics, and these pathways represent excellent targets for therapeutic intervention. One such regulatory pathway in RCC is the mTOR pathway, which interfaces with Akt (protein kinase B) and thePTEN tumor suppressor gene (Barthelemy et al., 2013;Hudes, 2009). Expression of mTOR is upregulated by various growth factors or by mutation or loss ofPTEN. Through complex pathways involving a variety of intermediaries, the mTOR pathway leads to increased expression of HIFs and other growth-promoting and potentially tumorigenic sequelae.Inhibition of mTOR with temsirolimus (Torisel) has yielded prolonged survival in patients with poor-risk, metastatic RCC, and everolimus (Afinitor) has shown efficacy for patients in whom tyrosine kinase inhibitors have failed, confirming the clinical relevance of the mTOR pathway (seeFig. 97.7) (Hudes et al., 2007;Motzer et al., 2008). Both of these mTOR inhibitors are now also approved by the FDA.

Proliferative index, as defined by proliferating cell nuclear antigen or Ki-67 staining, has correlated with pathologic parameters and clinical outcomes in RCC, suggesting that regulation of the cell cycle plays an important role in the tumor biology of RCC (Bui et al., 2004;Tollefson et al., 2007). Increased expression of transforming growth factor-α and its receptor tyrosine kinase, the epidermal growth factor receptor (EGFR), have been reported in RCC and may contribute to tumorigenesis by promoting cellular proliferation or transformation through an autocrine mechanism. Unfortunately, phase II clinical trials using agents that target EGFR, including erlotinib (Tarceva), gefitinib (Iressa), panitumumab (Vectibix), and lapatinib (Tykerb), demonstrated a lack of substantial activity in patients with advanced RCC (Rini, 2010). Based on these results, agents targeting the EGFR pathway have fallen out of favor for RCC, although selective treatment of patients who overexpress EGFR may still be a consideration within precision medicine trials.

The hepatocyte growth factor and its receptor, the c-MET proto-oncogene, may also contribute to the pathogenesis of RCC (Gibney et al., 2013;Giubellino et al., 2009;Harshman and Choueiri, 2013). The role of activating mutations of the c-MET proto-oncogene in the cause of hereditary papillary RCC has already been discussed, but data suggest that upregulated expression of this ligand may also occur in most of the histologic subtypes of RCC (Giubellino et al., 2009;Harshman and Choueiri, 2013). Hepatocyte growth factor is expressed by normal proximal tubular cells, where it is involved in branching tubulogenesis of the developing kidney and regeneration after renal injury. Increased serum levels of hepatocyte growth factor have also been reported in most patients with RCC, independent of histologic subtype, and activation of the receptor by phosphorylation at two sites is associated with cancer progression, making c-Met a potential therapeutic target for papillary and other forms of RCC (Gibney et al., 2013). Cabozantinib, an inhibitor of VEGFR, MET, and AXL, demonstrated a significant clinical benefit versus sunitinib, which primarily targets VEGFR, as first-line treatment for intermediate- to poor-risk RCC, suggesting a potential superiority to dual pathway inhibition (Choueiri et al., 2017a).

Signal transduction refers to all biochemical processes by which cells translate extracellular signals originating from their environment into specific responses. During the past 50 years, intensive research uncovered the enzymes and molecules that participate in this process (i.e., receptors, second messengers, phospholipases, kinases, phosphatases, etc.) and delineated the mechanisms by which cells integrate multiple signals. Signal transduction is now thought to occur through tightly organized networks in which protein–protein interactions and reversible assembly of signaling complexes are controlled by a small number of modular domains. The challenge in the future will be to better understand how signal specificity is achieved. Such information is vital to the reawakening of ‘systems biology’. Signal transduction is critical in regulating the lung nonrespiratory functions. Secretion of mucins by bronchial epithelial cells and surfactant by type II alveolar epithelial cells, phagocytosis and the respiratory burst by alveolar macrophages, production of cytokines by various cells, replacement of various cell types by cell division, and differentiation are among processes that are highly regulated through signaling pathways in the lung. Aberrant signal transduction underlies pathological changes such as fibroblast proliferation and cancer.

Intracellular Signal Transduction Mechanism of Opioid Receptors

The opioid receptors belong to the G-protein-coupled receptor family. It has been demonstrated that activation of the opioid receptors leads to activation of the pertussis toxin-sensitive G proteins (Gi or Go or both). Expression of the cloned opioid receptors in cultured cells by transfection of the cloned cDNAs has facilitated analysis of the intracellular signal transduction mechanisms activated by the opioid receptors (Fig. 24.6).2 Adenylate cyclase is inhibited by opioid receptor activation, with a resulting reduction of the cellular cyclic adenosine monophosphate (AMP) content. Electrophysiologically, the voltage-gated Ca2+channel is inhibited and the inwardly rectifying potassium (K+) channels are activated by the opioid receptors. As a result, neuronal excitability is reduced by activation of the opioid receptors. However, the role of adenylate cyclase in opioid receptor activation is complex. For example, long-term tolerance to opioids has been thought to be associated with superactivation of adenylyl cyclase activity, which is a counterregulatory response to the decrease in cyclic AMP levels seen after acute opioid administration.36 That this effect is prevented by pretreatment of cells with pertussis toxin demonstrates involvement of G proteins (Gi or Go or both).

Beyond adenylate cyclase, other regulatory components participate in the coupling of opioid receptor binding and a cellular response. It was shown that extracellular signal-related kinase, a class of mitogen-activated protein kinases, is activated by the opioid receptors.37 Opioid-induced activation of extracellular signal-related kinase can lead to increase in arachidonate release37 and expression of immediate early genes, c-fos and junB.38

Chronic exposure of the opioid receptors to agonists induces cellular adaptation mechanisms, which may be involved in opioid tolerance, dependence, and withdrawal symptoms. Several investigators have shown that short-term desensitization probably involves phosphorylation of the opioid receptors through protein kinase C.39 A number of other kinases also have been implicated, including protein kinase A and β-adrenergic receptor kinase (BARK), a member of the G-protein-coupled receptor kinase (GRK).40 BARKs selectively phosphorylate agonist-bound receptors and thereby promote interactions with β-arrestins, which interfere with G-protein coupling and promote receptor internalization. β-Arrestin 2 functions as a scaffolding protein that interacts with signal transducers, and the recruitment of β-arrestin 2 induced by opioid receptor activation is involved in the regulation of activity of c-Src, Akt, and mitogen-activated protein kinases (Fig. 24.7).41 The c-Src inhibitor, dasatinib, attenuated and reversed morphine-induced tolerance in mice, suggesting that c-Src recruited by β-arrestin 2 is involved in the morphine-induced tolerance.42 Acute morphine-induced analgesia was enhanced in mice lacking β-arrestin 2, suggesting that this protein contributes to regulation of responsivity to opioidsin vivo.43 Therefore, β-arrestin modification by associated kinases serves a critical role in the coupling between agonist binding to opioid receptors and their ability to develop and sustain an analgesic response.

PRINCIPLES OF CELLULAR SIGNALING

Signal transduction or cell signaling concerns the mechanisms by which biological information is transferred between cells. Functional coordination in complex multicellular organisms requires intercellular communication between a diverse range of specialized cell types in various tissues and organs. Maintaining this coordination requires a constant and dynamic stream of intercellular communication. Adjacent cells can communicate directly by interactions of surface proteins, and through specialized plasma membrane junctions (gap junctions) that allow the direct passage of small cytoplasmic molecules from one cell to the other. Long range cell-to-cell communication is possible through the involvement of extracellular signaling molecules (such as hormones and neurotransmitters) that are synthesized and released by specific cells, diffuse or circulate to target cells, and elicit specific responses in target cells that express receptors for the particular signal. The responses to the extracellular signal are generated by diverse signal transduction mechanisms that frequently involve small intracellular molecules (second messengers) that transmit signals from activated receptors to the cell interior, resulting in changes in the expression of genes and the activity of enzymes. These intercellular and intracellular signaling pathways are essential to the growth, development, metabolism, and behavior of the organism.

At the level of individual cells, signaling is crucial in division, differentiation, metabolic control and death. Cell signaling pathways are involved in the pathophysiology of many diseases. Cancer is a disease of signaling malfunction due to inactivation of a growth-inhibiting (tumor suppressor) pathway, or to activation of a growth-promoting (oncogene) pathway by genetic mutation. Diabetes results from defects in insulin signaling involved in blood glucose homeostasis. Cell signaling pathways are also involved in the mechanisms of action of many drugs, including local and general anesthetics. Knowledge of basic cell signaling mechanisms is therefore essential for an understanding of many pathophysiologic and pharmacologic mechanisms. Progress in this area has been enhanced by the completion of a draft sequence of the human genome, which includes at least 3775 genes (∼14% of all genes) involved in signal transduction. The new challenge in cell signaling is to understand the temporal and spatial regulation of signaling events in cells.

Signal Transduction–Related Oncogenes

Intermediate steps that effectively translate ligand-receptor binding to an intracellular signal are essential in mediating functional responses of the cell. Mutations in genes that encode key proteins that participate in signal transduction can also lead to cellular transformation (Fig. 1.5). In this regard, the largest family of oncogenes encodes proteins with protein kinase activity. Many members of this large oncogene group are expressed by neoplasms of the GI tract, and these include the Src nonreceptor tyrosine kinase that associates with the inner surface of the plasma membrane.

G proteins regulate signaling of the large family of GPCRs through the exchange of guanosine triphosphate with guanosine diphosphate. In this regard, theras family of genes, which encodes a family of proteins related to the G proteins, are among the most commonly detected oncogenes in GI tract cancers. Theras family contains 3 genes:H-ras, K-ras, andN-ras. These factors are essential to transduce signals from various growth receptor signaling cascades and point mutations that result in activating amino acid substitutions at critical hot spot positions convert the normal gene into an oncogene.

To date, almost allras mutations in GI malignancies occur in theK-ras oncogene. The highest mutation frequency is found in tumors of the exocrine pancreas (>90%).33Ras genes activated through point mutation have been identified in approximately 50% of colonic cancers as well as a subset of serrated tumors (seeFig. 1.4).34

Most oncogenic mutations inras cause biochemical changes that maintain it in the active, guanosine triphosphate–bound state by reducing guanosine triphosphatase activity or by destabilizing the inactive guanosine diphosphate–bound form. However, severalras mutants retain significant guanosine triphosphatase activity; therefore, other mechanisms that convertras to a transforming protein may be involved.35

A functional consequence of ras activation is the phosphorylation and activation of key downstream serine/threonine kinases. One important target of ras is B-raf. In colon cancers without an identifiableK-ras mutation, 20% possess an activatingB-raf mutation,36 consistent with the concept that activation of an oncogenic pathway can be achieved through an alteration in any of several sequential components of a particular pathway (seeFig. 1.5).

Lipid Signaling

The elucidation of cell signaling mechanisms and the variety of molecules that are employed in these myriad of processes is particularly well exemplified by the lipid messengers. Except for the abovementioned steroid hormones, lipids have long been thought to function mainly in energy metabolism and membrane structure. This last two decades or so has culminated in the broad recognition that membrane phospholipids provide many of the important cell signaling molecules via phospholipases and lipid kinases. Key is the role of phospholipase C in hydrolyzing phosphatidylinositol bisphosphate (PIP2) to release diglyceride that activates protein kinase C (PKC) and inositol triphosphate (IP3), which mobilizes intracellular Ca2+, central to so many regulatory processes (see Section IIC). The phosphorylation of PIP2 at the three-position to produce PIP3 promotes vesicular trafficking and other cellular processes. Phospholipase D releases phosphatidic acid, and phospholipase A2 provides arachidonic acid, which is converted into prostaglandins, leukotrienes, lipoxins, and various P450 products; these ligands in turn bind to unique families of receptors, as does platelet activating factor (PAF). The more recent recognition in the last decade of the importance of sphingolipids and ceramide in signaling and the discoveries of the unique lysophosphatidic acid and sphingosine phosphate families of receptors has sparked the search for other new receptors for lipids. It is clear that the search for new lipid second messengers and their receptors and functions will continue unabated into the future. The newly emerging field of lipidomics (see www.lipidmaps.org) holds the promise of expanding our ability to interrogate in greater detail the specificity of agonists and receptors and their effects on lipid signaling events [18].

Signaling Transduces One Event into Another

In its broadest context, cell signaling involves the transduction of some event into another event. In sensory transduction, a sensory cell is exposed to some external signal that is transduced to produce a nervous signal, the action potential. As we will see later in Chapter 3.2, this action potential can move along cell membranes to rapidly convey the signal, the action potential, to remote parts of the sensory neuron. The action potential is then transduced to release neurotransmitter at the synapse—the gap between one neuron and another. The neurotransmitter is then transduced to form the response of the postsynaptic cell, the one on the other side of the synapse. In the case of cutaneous (skin) senses, the original sensory signal is mechanical—a push or a pull on the nerves in the skin. The mechanical signal is transduced to an electrical signal, and the electrical signal is then transduced to a chemical signal. This simple series of events illustrates the use of mechanical, electrical, and chemical signals in the body (see Figure 2.8.1).

Signal Transduction, Cellular Communication, and Perception of the Environment

Communication between cells in multicellular organisms is essential because this makes it possible for individual cells to coordinate their activities. This communication is typically carried out by extracellular messenger molecules, including hormones, growth and differentiation factors, interleukins, and neurotransmitters. These extracellular messengers are most often detected by receptors that are present on the surface of the responding cell. Signal transduction is the process in which binding of an extracellular messenger to the cell surface receptor is translated into changes in biochemistry, cell biology, and gene transcription that make it possible for the cell to respond to the information that was received.

Many aspects of multicellular life are regulated by or involve extracellular messenger molecules, including cell growth, cell division, cell death, differentiation, cell migration, metabolism, the immune response, and neuronal communication. In addition, signal transduction is essential for processes such as vision and smell that are used by multicellular organisms to perceive their environment. Finally, unicellular organisms depend on cell surface receptors and signal transduction to detect chemical cues related to the presence of mates, competitors, predators, noxious substances, and food.

Multicellular organisms use a large variety of molecules to carry information between cells, including amino acids, amino acid derivatives, peptides, proteins, steroids, prostaglandins, nucleosides, and nucleotides. Several types of receptors have evolved to detect these messengers, including G protein-coupled receptors, receptor protein-tyrosine kinases, receptor serine/threonine kinases, protein-tyrosine phosphatases, ion channels, and transcription factors. In addition, there are receptors that do not have any intrinsic biochemical activity; these include B-cell receptors, T-cell receptors, integrins, interleukin receptors, and others. These receptors typically cooperate with other proteins that contribute to the activities needed for signal transduction to occur. To illustrate the complex nature of this process, descriptions of signal transduction by G protein-coupled receptors and receptor protein-tyrosine kinases have been included below.

G-Protein–Coupled Receptors

G-protein–coupled receptors (GPCRs) respond to extracellular stimuli, like hormones, by interaction with a G-protein, transducing a signal across the membrane into the cellular interior. After GPCR activation, Gα-subunits bind GTP and become active, further activating downstream signaling factors like the enzyme adenylyl cyclase (AC), which synthesizes cyclic-AMP (cAMP). Activated G-proteins interact with downstream signaling factors to alter the production of second messengers such as inositolphosphates, calcium, and cAMP. GPCRs that activate the Gi class of Gα subunits inhibit cAMP production and GPCRs that activate the Gs class of Gα subunits activate cAMP production. cAMP, in turn, activates the cAMP-dependent protein kinase, protein kinase A (PKA). The PKA activation pathway is an example of a signal transduction cascade, in which tying several signaling events together amplifies the original signal inside the cell. For each activated GPCR molecule, many G-proteins can be activated, and each active G-protein can synthesize many cAMP molecules, continuing the cascade to PKA and further downstream.

GPCRs are highly involved in pituicyte functions. Impaired signaling was demonstrated in pituitary adenomas. Gsα point mutations have been demonstrated in GH-secreting pituitary tumors [6].

Pertuit et al. [7] have widely analyzed Gsα alterations in GH-secreting adenomas.

The only mutations so far unequivocally identified and observed in 30–40% of the GH-secreting tumors concern the gsp oncogene. Despite the large individual variations of Gsα mRNAs [7], the level of Gsα proteins is always lower in gsp positive (gsp+) compared to gsp negative (gsp−) tumors [7,8]. It has been previously suggested that activation of Gsα induces a conformational change that prevents its attachment to membranes and increases its degradation rate, which could involve the proteasome [9].

In addition to the gsp oncogene, an overexpression of the WtGsα protein has been observed in a subset of gsp− adenomas. Approximately 60% of these gsp− tumors express high levels of Gsα compared to normal human pituitary cells. The GNAS locus (guanine nucleotide binding protein (G protein), alpha stimulating activity polypeptide 1), which maps on human chromosome 20q13, consists of a complex region with multiple alternative spliced transcripts encoding multiple protein products. In most human tissues, Gsα is biallelically expressed but, in specific tissues, Gsα is imprinted [10,11]. In pituitary tumours, Gsα-encoding transcripts are monoallelically expressed, predominantly from the maternal allele [12]. In almost all cases of gsp+ somatotroph adenomas, the GNAS-activating mutation occurs on the active maternal allele [12]. It is well known that genomic imprinting dysregulations can impact gene expression levels and so can participate in tumorigenesis. A strong imprinting relaxation, with a paternally derived expression of Gsα, has been found only in gsp− tumors. Thus, other mechanisms that could account for WtGsα overexpression remain to be identified.

Gs proteins couple hormonal stimulation of various cell-surface receptors to the activation of AC [7]. AC activation leads to the generation of intracellular second-messenger cAMP, which stimulates the PKA, the main cAMP effector. The phosphodiesterases (PDEs) contribute to the complexicity and specificity of the cAMP pathway by hydrolyzing cAMP. It is now well established that cAMP is compartmentalized in cells. In response to an elevation of cAMP, PDEs can be activated directly by PKA (i.e., rapid feedback regulation) and/or by induction of PDE gene transcription (i.e., long-term regulation) [13]. Thus, the spatiotemporal balance between PKA and PDE activities is a determinant in the control of the cAMP signaling.

In the absence of PDE inhibitors, no difference in intracellular cAMP levels between gsp+ and gsp− adenomas was detectable [7]. Persani et al. [14] demonstrated that the transcripts of PDE4C and 4D, as well as those of PDE8, were overexpressed in gsp+ tumors [13], which were correlated with a sevenfold increase in PDE activity. The two nuclear proteins, the CREB protein and the inducible cAMP early repressor (ICER), are the main and best-characterized final targets of cAMP. The mRNA levels of the transcription factors CREB and ICER are both increased in the gsp+ tumors [15]. The phosphorylated CREB levels are similar in the two types of tumors, although PDE blockade induces an increase in P-CREB (PhosphoCREB) in gsp+ tumors. These results suggest that an increase in PDE activity could counteract the activation of the cAMP pathway and may have an impact on the phenotype of the gsp+ tumors [13].

Besides alterations of the cAMP pathway in gsp+ tumors, several lines of evidence also suggest the existence of cAMP pathway alterations in GH-secreting adenomas overexpressing WtGsα. Relatively high levels of CREB or ICER mRNAs have been observed in some gsp− tumors [15].

The overexpression of WtGsα enhances intracellular cAMP accumulation and stimulates the cAMP pathway (P-CREB level). An increase in CREB-dependent transcription is also observed both in the presence of the gsp oncogene and with the overexpression of WtGsα in GH3 cells [7].

In order to accurately determine the role of Gsα alterations in the initiation and progression of the GH-secreting adenomas, Pertuit et al. [7] realized a study on pituitary cells, finding that the induction of the expression of the gsp oncogene initiates a considerable increase in the AC activity, which is associated with an increase in the intracellular cAMP level. A weak but long-lasting activation of the AC, associated with a slight increase in the cAMP level, is also observed in response to overexpression of WtGsα. cAMP progressively decreases despite continuous transgene expression, suggesting a potential involvement of the PDEs. This may represent a second mechanism of feedback in addition to the posttranscriptional regulation of the gsp oncogene [7,16].

These mutations inhibit Gsα GTPase activity, resulting in GHRH ligand-independent constitutive activation of cAMP, which results in GH-transcriptional activation and somatotroph proliferation via a CREB in the GH promoter [2].

Significantly, higher amounts of Ser133phosphorylated, and hence activated CREB, have been reported in some GH-secreting pituitary tumors compared with the levels found in nonfunctional (NF) tumors. This augmented CREB activity was evident even in tumors that did not manifest a Gsα mutation. This would suggest that CREB activation may occur via a Gs-independent mechanism [2]. It is possible that the stimulatory/inhibitory polypeptides and steroid hormones released by hypothalamus and peripheral endocrine organs could alter pituitary gene expression and hormone secretion [2].

10.3.4 Significantly enriched signaling pathways of group N: Having negative correlation with ZMI in the wound-healing process

The following signaling pathways are significantly enriched by the genes in group N (Fig. 10.A7 and Table 10.A4 in Appendix). These neurotransmitter-related signaling pathways have appeared to be inhibited during the wound-healing process. They may not be as crucial for regenerability as the abovementioned pathways, but they support the functional recovery of neural transmission in the cerebellar wound-healing process.

Table 10.A4. The significantly enriched pathways in group N [493].

Some examples of these neurotransmitter-related signaling pathways are Beta1 adrenergic receptor signaling pathway, Beta2 adrenergic receptor signaling pathway, synaptic vesicle trafficking, muscarinic acetylcholine receptor 2 and 4 signaling pathway, metabotropic glutamate receptor group II signaling pathway, 5-HT1 type receptor-mediated signaling pathway, metabotropic glutamate receptor group III signaling pathway, Enkephalin release pathway, GABA-B receptor II signaling pathway, Beta3 adrenergic receptor signaling pathway, dopamine receptor-mediated signaling pathway, opioid prodynorphin pathway, opioid proenkephalin pathway, 5-HT4 type receptor-mediated signaling pathway, muscarinic acetylcholine receptor 1 and 3 signaling pathway, ionotropic glutamate receptor signaling pathway, and opioid proopiomelanocortin pathway. These signaling pathways are all related to neurotransmitters and receptors in neural transmission and their roles in the restoration and neurogenesis of the cerebellar wound-healing process of zebrafish need further elaboration.