Signaling by Chemokine Receptors

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Signaling by Chemokine Receptors

The expression of chemokine receptors by a variety of cell types within the CNS and the significance of their expression in disease and physiology has led to great interest in the molecular mechanisms by which chemokine-mediated responses are induced. To that end, a number of the investigative efforts on chemokines have focused on intracellular signaling. Data from studies indicate that stimulation of chemokine receptors results in the activation of a variety of effector molecules. Many of these are well-established targets of GPCR signaling, but some, like Jak/STAT molecules, are being recognized for the first time. The following section will present a brief review of the current knowledge of chemokine receptor signaling and their potential implications for glioma biology.

As noted previously, chemokine receptors are GPCRs. And while there is evidence suggesting that they can couple to G proteins other than Gi, many of the studies to date have described chemokine-induced signaling events in the context of what is already known about receptors coupled to Gi. For example, in primary mouse astrocytes treated with RANTES, a reduction in cAMP levels and PKA activity is observed. This event appears to be a dose-dependent one and very likely results from the inhibition of AC by the αi subunit of the G protein heterotrimeric complex. Reduced PKA activity may be relevant to gliomagenesis, as upregulated PKA activity in A 172 cells (a human malignant glioma cell line) is associated with reduced proliferation, differentiation, and increased apoptosis. Thus, RANTES-mediated reduction in PKA activity could serve to disinhibit proliferation, promote dedifferentiation and reduce apoptotic death, all of which could contribute to gliomagenesis.

Focus on the βγ dimer as a major component of GPCR signaling has led to the identification of “signature” intracellular events that follow chemokine receptor activation. For example, the mobilization of intracellular calcium is considered an indication of a functional chemokine receptor. The rise in [Ca2+]i occurs as a result of Ca2+ release from intracellular stores, an event mediated by the IP3 generated from cleavage of PIP2 by PLCβ. Studies of PLCβ activation by GPCRs have demonstrated that it may be mediated by both the βγ dimer of Gi/o proteins and by the α monomer of Gq proteins. One report has suggested that the rise in [Ca2+]i following chemokine receptor activation may result from the influx of Ca2+ from the extracellular environment, based on the observation that the mobilization of Ca2+ in CCR3-expressing microglia provoked by RANTES exhibited sensitivity to nifedipine, a dihydropyridine derivative known to inhibit L-type calcium channels. However, given the absence of a voltage-dependent ion flux in microglial cells, it is not clear whether this is indeed the case.

A rise in [Ca2+]i has several consequences for intracellular signaling, because as a second messenger, Ca2+ plays a crucial role in the activation of effector molecules. Several reports have demonstrated that this principle holds true in chemokine-induced signaling. For example, Ca2+-sensitive potassium channels (KCa) in human macrophages are activated in the presence of both MIP-1β and SDF-1α. Another example is proline-rich tyrosine kinase 2 (Pyk2 or related adhesion focal tyrosine kinase (RAFTK)), a Ca2+-sensitive molecule activated in T cells in the presence of both RANTES and MIP-1β. A similar observation has been made of B cells in the presence of SDF-1α. A third example is PKC, the “classical” isoforms of which are sensitive to Ca2+. Treatment of hematopoietic progenitor cells with SDF-1α indicated the activation of PKC, as GF109203X, a pharmacological inhibitor that exhibits selectivity for some classical isoforms of PKC was able to reduce SDF-1α induced migration in these cells.

SDF-1α induced activation of PKC presents an interesting link to brain tumors because SDF-1α and its receptor have been implicated in brain tumor growth and development, and PKC expression and activity also has been reported to promote the growth of malignant gliomas. The level of PKC expression in neoplastie astrocytes is high compared to that of non-neoplastic astrocytes, and correlates with their basal rate of proliferation. Evidence in support of PKC activity as a cause rather than an effect of increased proliferation is persuasive, as treatment of neoplastic astrocytes with growth factors such as EGF that results in activation of PKC also stimulates proliferation. In addition, the use of pharmacological agents that directly activate PKC increase the rate of DNA synthesis. Conversely, pharmacological inhibition of PKC results in reduced DNA synthesis, decreased proliferation and irreversible growth arrest. Indeed, SDF-1α induced proliferation of glioma cells may be a function of PKC activation.

Given the role of PI3-K in cell migration and the data indicating that activation of PI3-K is observed in the presence of SDF-1α, it is not unreasonable to consider that SDF-1α may also contribute to the invasive behavior of brain tumors. As noted previously, SDF-1α has been reported to serve as a chemotactic factor for glioma cells, and expression of SDF-1α and CXCR4 has been observed to increase with tumor grade. One of the signature features of brain tumors is their diffuse infiltration of adjacent and distant brain structures. While it is possible that a high rate of proliferation may be able to explain infiltration of adjacent structures, it does not address how infiltration of distant brain structures could be achieved. SDF-1α, perhaps as a result of its ability to activate PI3-K and the role played by PI3-K in mediating cell migration, may be able to account in part for how such a phenomenon could be achieved.

Activation of PI3-K by SDF-1α also may be relevant to gliomagenesis in another regard. A consequence of PI3-K activity is the production of PIP3 . The production of PIP3 results in the recuitment and activation of protein kinase B (PKB or Akt), a serine/threonine kinase whose activation is strongly correlated with cell survival. Persistent activation of PKB is thus recognized as one mechanism used by transformed cells to resist cell death. Interestingly, a number of studies have reported that the level of PKB activity is relatively high in a variety of glioblastoma cell lines. As noted previously, overexpression of SDF-1α and CXCR4 in resected high grade brain tumor tissue is observed. Thus, SDF-1α also may contribute to gliomagenesis by inducing the persistent activation of PKB by positively regulating activation of PI3-K.

Recent evidence has provided further support for the idea that SDF-1α can promote the activation of PKB. Full activation of PKB requires phosphorylation at Thr308 and Ser473, and while it is widely accepted that phosphorylation at Thr308 is mediated by phosphatidylinositol dependent kinase-1 (PDK1), the identity of the second kinase, termed phosphatidylinositol dependent kinase-2 (PDK2), is unknown. Interestingly, it has been reported that PKCα, a Ca2+-sensitive isoform of PKC that is activated by SDF-1α, may serve as PDK2 in rat fad pad endothelial cells. Offered in support of this idea was data indicating that overexpression of PKCα correlated with increased phosphorylation of PKB at Ser473, while inhibition of PKCα activity was associated with reduced phosphorylation of PKB at Ser473. It was also demonstrated that PKCα could directly phosphorylate PKB at Ser473. These findings suggest that the contribution of SDF-1α in regulating activation of PKB may occur at two levels. One, upstream of PKB through activation of PI3-K and two, at the level of PKB by PKCα mediated phosphorylation of Ser473.

In addition to activating the proteins described above, chemokine receptor stimulation has also been reported to result in the activation of m itogen-activated protein kinases (MAPKs), evolutionarily conserved proteins that are intimately associated with cell proliferation, cell differentiation and cell death. The MAPK family currently is composed of 12 members that are divided among three groups: extracellular signal-regulated kinases (ERKs), jun-N-terminal kinases (JNKs), and p38s. The ERK group is composed of five members, ERKl, ERK2, ERK3, ERKS and ERK7. The JNK family is composed of three members, JNKl, JNK2 and JNK3. And the p38 family is composed of four members, p38α, p38β, p38γ and p38δ.

Activation of MAPKs is the result of a series of three stepwise signaling events that begins with the activated MAPK kinase kinase (MAPK3), a serine/threonine kinase which phosphorylates and thus activates a MAPK kinase (MAPK2). MAPK2s are dual specificity kinases (e.g. tyrosine and threonine) that phosphorylate and activate MAPKs. MAPKs are serine/threonine kinases that phosphorylate key enzymes and nuclear proteins, ultimately resulting in gene expression. This three-tiered architecture constitutes what is known as the MAPK signaling cascade.

Numerous studies have reported that phosphorylation of MAPK family members occurs following stimulation of chemokine receptors. For example, activation of ERK 1/2 is observed in the presence of chemokines that activate CXCRI, CXCR2, CXCR3, CXCR4, CCRl, CCR2, CCR3, CCR4, CCR5, and CX3CR1. Stimulation of CCRl, CCR3, CCR5 in immature dendritic cells also results in the activation of JNK and p38. Similar results are observed in human umbilical vein endothelial cells (HUVECs) following stimulation of CCR2. In the human T cell line, PM 1, the activation of the MAPK family member, p38, is observed following stimulation of CCR5. However, in hippocampal neurons, only activation of ERK 1/2 is observed following stimulation of CX3CR1 and CCR4. These results suggest that chemokine-induced activation of MAPK family members depends on a variety of factors, including cell type and chemokine receptor.

There is evidence indicating that chemokine-induced activation of ERK 1/2 results from activation of the MAPK signaling cascade. Raf-1, a 74 kDa MAPK3, is activated in primary neonatal mouse astrocytes following stimulation of CCRl and CCR5 with RANTES. MEK, a MAPK2 immediately upstream of ERK 1/2, is also activated in the presence of RANTES. In addition, dephosphorylation of Raf-1 at Ser259, an activating event, also occurs after stimulation of CCRl and CCR5. Furthermore, treatment with a Raf-1 inhibitor can block RANTES-induced gene expression in a dose-dependent manner. These results suggest that in primary neonatal mouse astrocytes, RANTES-induced activation of ERK 1/2 results from activation of the MAPK signaling cascade. Interestingly, it has been reported that phosphorylation of Raf-1 at Ser259 (an inhibitory mechanism) is mediated by PKA. As noted previously, RANTES treatment of astrocytes results in a reduction of PKA activity. Thus, RANTES-mediated reduction in PKA activity may contribute to increased Raf-1 activity by promoting conditions that would disfavor the deactivating phosphorylation of Raf-1 at Ser259.

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