Johan Lennartsson

Stem Cell Factor Receptor/c-Kit: From Basic Science to Clinical Implications

Stem cell factor (SCF) is a dimeric molecule that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Activation of c-Kit leads to its autophosphorylation and initiation of signal transduction. Signaling proteins are recruited to activated c-Kit by certain interaction domains (e.g., SH2 and PTB) that specifically bind to phosphorylated tyrosine residues in the intracellular region of c-Kit. Activation of c-Kit signaling has been found to mediate cell survival, migration, and proliferation depending on the cell type. Signaling from c-Kit is crucial for normal hematopoiesis, pigmentation, fertility, gut movement, and some aspects of the nervous system. Deregulated c-Kit kinase activity has been found in a number of pathological conditions, including cancer and allergy. The observation that gain-of-function mutations in c-Kit can promote tumor formation and progression has stimulated the development of therapeutics agents targeting this receptor, e.g., the clinically used inhibitor imatinib mesylate. Also other clinically used multiselective kinase inhibitors, for instance, sorafenib and sunitinib, have c-Kit included in their range of targets. Furthermore, loss-of-function mutations in c-Kit have been observed and shown to give rise to a condition called piebaldism. This review provides a summary of our current knowledge regarding structural and functional aspects of c-Kit signaling both under normal and pathological conditions, as well as advances in the development of low-molecular-weight molecules inhibiting c-Kit function.

Normal and Oncogenic Forms of the Receptor Tyrosine Kinase Kit

Kit is a receptor tyrosine kinase (RTK) that binds stem cell factor. This receptor ligand combination is important for normal hematopoiesis, as well as pigmentation, gut function, and reproduction. Structurally, Kit has both an extracellular and intracellular region. Theintra‐cellular region is comprised of a juxtamembrane domain (JMD), a kinase domain, a kinase insert, and a carboxyl tail. Inappropriate expression or activation of Kit is associated with a variety of diseases in humans. Activating mutations in Kit have been identified primarily in the JMD and the second part of the kinase domain and have been associated with gastrointestinal stromal cell tumors and mastocytosis, respectively. There are also reports of activating mutations in some forms of germ cell tumors and core binding factor leukemias. Since the cloning of the Kit ligand in the early 1990s, there has been an explosion of information relating to the mechanism of action of normal forms of Kit as well as activated mutants. This is important because understanding this RTK at the biochemical level could assist in the development of therapeutics to treat primary and secondary defects in the tissues that require Kit. Furthermore, understanding the mechanisms mediating transformation of cells by activated Kit mutants will help in the design of interventions for human disease associated with these mutations. The objective of this review is to summarize what is known about normal and oncogenic forms of Kit. We will place particular emphasis on recent developments in understanding the mechanisms of action of normal and activated forms of this RTK and its association with human disease, particularly in hematopoietic cells.

A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse

In horses, graying with age is an autosomal dominant trait associated with a high incidence of melanoma and vitiligo-like depigmentation. Here we show that the Gray phenotype is caused by a 4.6-kb duplication in intron 6 of STX17 (syntaxin-17) that constitutes a cis-acting regulatory mutation. Both STX17 and the neighboring NR4A3 gene are overexpressed in melanomas from Gray horses. Gray horses carrying a loss-of-function mutation in ASIP (agouti signaling protein) had a higher incidence of melanoma, implying that increased melanocortin-1 receptor signaling promotes melanoma development in Gray horses. The Gray horse provides a notable example of how humans have cherry-picked mutations with favorable phenotypic effects in domestic animals.

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

In this report we show that Tyr568 and Tyr570 are phosphorylated in vivo in the Kit/stem cell factor receptor (Kit/SCFR) following ligand-stimulation. By mutation of Tyr568 and Tyr570 to phenylalanine residues and expression of the mutated receptors in porcine aortic endothelial (PAE) cells, we could demonstrate a loss of activation of members of the Src family of tyrosine kinases when Tyr568 was mutated, while mutation of Tyr570 only led to a minor decrease in activation of Src family members. Mutation of both tyrosine residues led to a complete loss of Src family kinase activation. Phosphorylation of the adapter protein Shc by growth factor receptors provides association sites for Grb2-Sos, thereby activating the Ras/MAP kinase pathway. A much lowered degree of Shc phosphorylation, Ras and Erk2 activation and c-fos induction was seen in the Y568F mutant, while in the Y570F mutant these responses were less affected. In contrast, the mitogenic response was only slightly reduced. In a mutant receptor with both Tyr568 and Tyr570 mutated to phenylalanine residues, no phosphorylation of Shc and no activation of Ras and Erk2 was seen in response to stem cell factor stimulation, very weak induction of c-fos was seen and the mitogenic response was severely depressed. These data show that Ras/MAP kinase activation and c-fos induction by Kit/SCFR are mediated by members of the Src family kinases. However, the mitogenic response is only to a minor extent dependent on Src kinase activity.

VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis

VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a “survival,” rather than an “angiogenic” factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.

Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors

The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.

VEGF-B inhibits apoptosis via VEGFR-1–mediated suppression of the expression of BH3-only protein genes in mice and rats

Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.

Identification of Tyr-703 and Tyr-936 as the primary association sites for Grb2 and Grb7 in the c-Kit/stem cell factor receptor.

In this paper we demonstrate the presence of two novel in vivo autophosphorylation sites in the c-Kit/stem cell factor receptor (c-Kit/SCFR): Tyr-703 in the kinase insert and Tyr-936 in the C-terminal tail. We furthermore demonstrate that the adapter protein Grb2 is a specific binding partner for both phosphorylated Tyr-703 and phosphorylated Tyr-936, whereas the adapter protein Grb7 binds selectively to phosphorylated Tyr-936. It is shown that the association occurs through the Src homology 2 (SH2) domains of Grb2 and Grb7. Binding of Grb2 to Tyr-703 in the c-Kit/SCFR provides a link to the Ras/mitogen-activated protein kinase pathway.

Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3β phosphorylation

Platelet-derived growth factor CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase 3β (GSK3β) activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-hydroxydopamine–induced Parkinson’s dopaminergic neuronal death, and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody, or short hairpin RNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3β phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC–PDGF receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions.

The Stem Cell Factor Receptor/c-Kit as a Drug Target in Cancer

Tyrosine phosphorylation has a key role in intracellular signaling. Inappropriate proliferation and survival cues in tumor cells often occur as a consequence of unregulated tyrosine kinase activity. Much of the current development of anti-cancer therapies tries to target causative proteins in a specific manner to minimize side-effects. One attractive group of target proteins is the kinases. c-Kit is a receptor tyrosine kinase that normally controls the function of primitive hematopoietic cells, melanocytes and germ cells. It has become clear that uncontrolled activity of c-Kit contributes to formation of an array of human tumors. The unregulated activity of c-Kit may be due to overexpression, autocrine loops or mutational activation. This makes c-Kit an excellent target for cancer therapies in these tumors. In this review we will highlight the current knowledge on the signal transduction molecules and pathways activated by c-Kit under normal conditions and in cancer cells, and the role of aberrant c-Kit signaling in cancer progression. Recent advances in the development of specific inhibitors interfering with these signal transduction pathways will be discussed.