The TAM Subfamily of Receptor Tyrosine Kinases: The Early Years.

The TAMs are a subfamily of receptor tyrosine kinases (RTKs) comprised of three members, Tyro3, Axl and Mer. Evidence in support of the existence of this subfamily emerged from a screen for novel RTKs performed in the laboratory of Dr. Greg Lemke in 1991. A PCR-based approach to selectively amplify tyrosine kinase-specific genes yielded 27 different tyrosine kinase genes, of which 13 were novel (the "Tyros"). Of these, Tyro3, 7 and 12 were more closely related to each other than to any other kinases and it was proposed that they constituted a novel subfamily of RTKs. Additional support for this hypothesis required determining the complete sequences for these receptor tyrosine kinases. By the end of 1991, full-length sequences for Tyro7 (Axl) revealed a unique extracellular domain organization that included two immunoglobulin-like domains and two fibronectin type III repeats. In 1994, the complete sequences for Tyro12 (Mer) and Tyro3 were shown to have an extracellular region domain structure similar to that of Axl. In 1995, Gas6 and Pros1 were reported as ligands for Tyro3 and Axl, setting the stage for functional studies. The Lemke lab and its many trainees have since played leading roles in elucidating the physiological relevance of the TAMs.

Neuregulins 1, 2, and 3 Promote Early Neurite Outgrowth in ErbB4-Expressing Cortical GABAergic Interneurons.

The neuregulins (Nrgs 1-4) are a family of signaling molecules that play diverse roles in the nervous system. Nrg1 has been implicated in the formation of synapses and in synaptic plasticity. Previous studies have shown Nrg1 can affect neurite outgrowth in several neuronal populations, while the role of Nrg2 and Nrg3 in this process has remained understudied. The Nrgs can bind and activate the ErbB4 receptor tyrosine kinase which is preferentially expressed in GABAergic interneurons in the rodent hippocampus and cerebral cortex. In the present study, we evaluated the effects of Nrgs 1, 2, and 3 on neurite outgrowth of dissociated rat cortical ErbB4-positive (+)/GABA+ interneurons in vitro. All three Nrgs were able to promote neurite outgrowth during the first 2 days in vitro, with increases detected for both the axon (116-120%) and other neurites (100-120%). Increases in the average number of primary and secondary neurites were also observed. Treatment with the Nrgs for an additional 3 days promoted an increase in axonal length (86-96%), with only minimal effects on the remaining neurites (8-13%). ErbB4 expression persisted throughout the dendritic arbor and cell soma at all stages examined, while its expression in the axon was transient and declined with cell maturation. ErbB4 overexpression in GABAergic neurons promoted neurite outgrowth, an effect that was potentiated by Nrg treatment. These results show that Nrgs 1, 2, and 3 are each capable of influencing dendritic and axonal growth at early developmental stages in GABAergic neurons grown in vitro.

MerTK inhibition in tumor leukocytes decreases tumor growth and metastasis.

MerTK, a receptor tyrosine kinase (RTK) of the TYRO3/AXL/MerTK family, is expressed in myeloid lineage cells in which it acts to suppress proinflammatory cytokines following ingestion of apoptotic material. Using syngeneic mouse models of breast cancer, melanoma, and colon cancer, we found that tumors grew slowly and were poorly metastatic in MerTK-/- mice. Transplantation of MerTK-/- bone marrow, but not wild-type bone marrow, into lethally irradiated MMTV-PyVmT mice (a model of metastatic breast cancer) decreased tumor growth and altered cytokine production by tumor CD11b+ cells. Although MerTK expression was not required for tumor infiltration by leukocytes, MerTK-/- leukocytes exhibited lower tumor cell-induced expression of wound healing cytokines, e.g., IL-10 and growth arrest-specific 6 (GAS6), and enhanced expression of acute inflammatory cytokines, e.g., IL-12 and IL-6. Intratumoral CD8+ T lymphocyte numbers were higher and lymphocyte proliferation was increased in tumor-bearing MerTK-/- mice compared with tumor-bearing wild-type mice. Antibody-mediated CD8+ T lymphocyte depletion restored tumor growth in MerTK-/- mice. These data demonstrate that MerTK signaling in tumor-associated CD11b+ leukocytes promotes tumor growth by dampening acute inflammatory cytokines while inducing wound healing cytokines. These results suggest that inhibition of MerTK in the tumor microenvironment may have clinical benefit, stimulating antitumor immune responses or enhancing immunotherapeutic strategies.

Cross-phosphorylation, signaling and proliferative functions of the Tyro3 and Axl receptors in Rat2 cells.

The dysregulation of receptor protein tyrosine kinase (RPTK) function can result in changes in cell proliferation, cell growth and metastasis leading to malignant transformation. Among RPTKs, the TAM receptor family composed of three members Tyro3, Axl, and Mer has been recognized to have a prominent role in cell transformation. In this study we analyzed the consequences of Tyro3 overexpression on cell proliferation, activation of signaling pathways and its functional interactions with Axl. Overexpression of Tyro3 in the Rat2 cell line that expresses Axl, but not Mer or Tyro3, resulted in a 5 fold increase in cell proliferation. This increase was partially blocked by inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway but not by inhibitors of the phosphatidylinositol 3-kinase (PI(3)K) signaling pathway. Consistent with these findings, an increase in ERK1/2 phosphorylation was detected with Tyro3 but not with Axl overexpression. In contrast, activation of Axl stimulated the PI(3)K pathway, which was mitigated by co-expression of Tyro3. The overexpression of Tyro3 enhanced Gas6-mediated Axl phosphorylation, which was not detected upon overexpression of a "kinase dead" form of Tyro3 (kdTyro3). In addition, the overexpression of Axl induced kdTyro3 phosphorylation. Co-immunoprecipitation experiments confirmed that the Axl and Tyro3 receptors are closely associated. These findings show that overexpression of Tyro3 in the presence of Axl promotes cell proliferation, and that co-expression of Axl and Tyro3 can affect the outcome of Gas6-initiated signaling. Furthermore, they demonstrate a functional interaction between the members of the TAM receptor family which can shed light on the molecular mechanisms underlying the functional consequences of TAM receptor activation in cell transformation, neural function, immune function, and reproductive function among others.

Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during experimental autoimmune encephalomyelitis.

BACKGROUND: Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays. RESULTS: Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals. CONCLUSIONS: These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.

Gas6 deficiency increases oligodendrocyte loss and microglial activation in response to cuprizone-induced demyelination.

The TAM family of receptor protein tyrosine kinases comprises three known members, namely Tyro3, Axl, and Mer. These receptors are widely expressed in the nervous system, including by oligodendrocytes, the cell type responsible for myelinating the CNS. We examined the potential role of the TAM family and of their principle cognate ligand, Gas6 (growth arrest gene 6), in modulating the phenotype of the cuprizone model of demyelination. We found that the expression profiles of Axl, Mer, and Gas6 mRNA were increased in the corpus callosum in a temporal profile correlating with the increased migration and proliferation of microglia/macrophages in this model. In contrast, expression of Tyro3 decreased, correlating with the loss of oligodendrocytes. Gas6 both promoted in vitro survival of oligodendrocytes (39.3 +/- 3.1 vs 11.8 +/- 2.4%) and modulated markers of activation in purified cultures of microglia (tumor necrosis factor alpha mRNA expression was reduced approximately 48%). In Gas6-/- mice subjected to cuprizone-challenge, demyelination was greater than in control mice, within the rostral region of the corpus callosum, as assessed by luxol fast blue staining (myelination reduced by 36%) and by ultrastructural analysis. An increased loss of Gst-pi (glutathione S-transferase-pi)-positive oligodendrocytes was also identified throughout the corpus callosum of Gas6-/- mice. Microglial marker expression (ionized calcium-binding adapter molecule 1) was increased in Gas6-/- mice but was restricted to the rostral corpus callosum. Therefore, TAM receptor activation and regulation can independently influence both oligodendrocyte survival and the microglial response after CNS damage.

Interleukin-6 produces neuronal loss in developing cerebellar granule neuron cultures.

CNS levels of the cytokine interleukin-6 (IL-6) are elevated during CNS injury and disease, but it is unclear if IL-6 contributes to the pathologic process. Our studies show that in a well-characterized CNS developmental model system, primary cultures of rodent cerebellar granule neurons, chronic exposure to IL-6 during neuronal development can result in cell damage and death in a subpopulation of developing granule neurons. Chronic exposure to IL-6 also increased the susceptibility of the granule neurons to a toxic insult produced by excessive activation of NMDA receptors. These results are consistent with a role for IL-6 in the neuropathology observed in the developing CNS during injury and disease.

Neuregulin-2 is synthesized by motor neurons and terminal Schwann cells and activates acetylcholine receptor transcription in muscle cells expressing ErbB4.

Acetylcholine receptor (AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Here, we show that motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin-2 is a candidate for a signal that regulates synaptic differentiation.

Gas6 binding to photoreceptor outer segments requires gamma-carboxyglutamic acid (Gla) and Ca(2+) and is required for OS phagocytosis by RPE cells in vitro.

The phagocytosis of photoreceptor outer segments by retinal pigment epithelial (RPE) cells plays a critical role in preserving normal retinal function. Recently the receptor protein tyrosine kinase (RTK) Mer, has been shown to be necessary for this cellular process to take place. Gas6, the ligand for the Mer RTK, can specifically and selectively stimulate the phagocytosis of photoreceptor outer segments (OS) by normal cultured rat RPE cells, as we have previously reported. The Gas6 protein has been shown to associate with plasma membrane phosphatidylserine by its amino-terminal portion, while its carboxyl-terminal portion can bind and activate Mer and its related RTKs, Axl and Tyro-3. Given the capability of Gas6 to interact with more than one molecule, we have performed a series of experiments to further dissect the interactions of Gas6 with the OS and RPE and to determine the specific calcium requirements necessary for Gas6 to exert its stimulatory effect on phagocytosis. These experiments show that Gas6 must bind to OS before the stimulation of OS ingestion can occur and that this binding requires a Ca(2+) concentration of 500-600 microM. The same Ca(2+) concentration is required for the Gas6 mediated stimulation of OS ingestion. We further demonstrate that in order to bind to OS and to stimulate OS phagocytosis, Gas6 requires gamma-carboxylation in a vitamin K-dependent reaction. By analogy with other systems, we propose that Ca(2+) mediates the linkage between the gamma-carboxyglutamic acid (Gla)-rich N-terminal region of Gas6 with phospholipids, presumably phosphatidylserine, in the plasma membrane of the OS. Only after this binding has occurred can Gas6 interact with receptor molecule(s) on the surface of the RPE, and activate RPE cell signaling pathways leading to OS ingestion. These studies further underscore the importance of Gas6 in the phagocytic function of the RPE and open new avenues of investigation to understand the molecular events mediated and triggered by Gas6, and its interaction with the OS and RPE.