The Krüppel-like family of transcription factors (KLFs) have been widely studied in proliferating cells, though very little is known about their role in post-mitotic cells, such as neurons. We have recently found that the KLFs play a role in regulating intrinsic axon growth ability in retinal ganglion cells (RGCs), a type of central nervous system (CNS) neuron. Previous KLF studies in other cell types suggest that there may be cell-type specific KLF expression patterns, and that their relative expression allows them to compete for binding sites, or to act redundantly to compensate for another’s function. With at least 15 of 17 KLF family members expressed in neurons, it will be important for us to determine how this complex family functions to regulate the intricate gene programs of axon growth and regeneration. By further characterizing the mechanisms of the KLF family in the nervous system, we may better understand how they regulate neurite growth and axon regeneration.

Neurons in the adult mammalian CNS decrease in intrinsic axon growth capacity during development in concert with changes in Krüppel-like transcription factors (KLFs). KLFs regulate axon growth in CNS neurons including retinal ganglion cells (RGCs). Here, we found that knock-down of KLF9, an axon growth suppressor that is normally upregulated 250-fold in RGC development, promotes long-distance optic nerve regeneration in adult rats of both sexes. We identified a novel binding partner, MAPK10/JNK3 kinase, and found that JNK3 (c-Jun N-terminal kinase 3) is critical for KLF9's axon-growth-suppressive activity. Interfering with a JNK3-binding domain or mutating two newly discovered serine phosphorylation acceptor sites, Ser106 and Ser110, effectively abolished KLF9's neurite growth suppression and promoted axon regeneration These findings demonstrate a novel, physiologic role for the interaction of KLF9 and JNK3 in regenerative failure in the optic nerve and suggest new therapeutic strategies to promote axon regeneration in the adult CNS. Injured CNS nerves fail to regenerate spontaneously. Promoting intrinsic axon growth capacity has been a major challenge in the field. Here, we demonstrate that knocking down Krüppel-like transcription factor 9 (KLF9) via shRNA promotes long-distance axon regeneration after optic nerve injury and uncover a novel and important KLF9-JNK3 interaction that contributes to axon growth suppression and regenerative failure These studies suggest potential therapeutic approaches to promote axon regeneration in injury and other degenerative diseases in the adult CNS.

PurposeAdult central nervous system (CNS) neurons are unable to regenerate their axons after injury. Krüppel-like transcription factor (KLF) family members regulate intrinsic axon growth ability in vitro and in vivo, but mechanisms downstream of these transcription factors are not known.MethodsPurified retinal ganglion cells (RGCs) were transduced to express exogenous KLF9, KLF16, KLF7, or KLF11; microarray analysis was used to identify downstream genes, which were screened for effects on axon growth. Dual-specificity phosphatase 14 (Dusp14) was further studied using genetic (siRNA, shRNA) and pharmacologic (PTP inhibitor IV) manipulation to assess effects on neurite length in vitro and survival and regeneration in vivo after optic nerve crush in rats and mice.ResultsBy screening genes regulated by KLFs in RGCs, we identified Dusp14 as a critical gene target limiting axon growth and regeneration downstream of KLF9's ability to suppress axon growth in RGCs. The KLF9-Dusp14 pathway inhibited activation of mitogen-activated protein kinases normally critical to neurotrophic signaling of RGC survival and axon elongation. Decreasing Dusp14 expression or disrupting its function in RGCs increased axon growth in vitro and promoted survival and optic nerve regeneration after optic nerve injury in vivo.ConclusionsThese results link intrinsic and extrinsic regulators of axon growth and suggest modulation of the KLF9-Dusp14 pathway as a potential approach to improve regeneration in the adult CNS after injury.

A n adequate immune response is the result of a fine balance between activating and inhibitory signals that promotes elimination of the invading agent while suppressing hyperresponsiveness that could damage the host. Many mechanisms exist to accomplish this task, including the expression of both activating and inhibitory receptors by immune cells. A group of inhibitory receptors are characterized by the presence of one or more ITIM in their cytoplasmic tail. After interaction with their ligands, the tyrosine residues in the ITIM are phosphorylated by Src family tyrosine kinases. These phosphorylated tyrosine residues serve as docking sites for phosphatases, such as Src homology 2 domain-containing protein tyrosine phosphatase (SHP) 4 -1, -2, and SHIP, which then become activated and initiate the propagation of the inhibitory signal (1, 2). The leukocyte associated inhibitory receptor (LAIR)-1, also known as CD305, possesses two ITIM in its cytoplasmic tail that mediate its inhibitory capacity through interaction with SHP-1, SHP-2, and/or C-terminal Src kinase (3-5). This receptor is expressed by all leukocytes, NK cells, T cells, B cells, dendritic cells, monocytes, neutrophils, and hematopoietic stem cells (3, 6 -9). The ligands for LAIR-1, and for the highly homologous, secreted protein LAIR-2, are collagens, the most abundant type of protein in the body (10, 11). The interaction between LAIR-1 and collagens is of high affinity and is dependent on the conserved glycine-proline-hydroxyproline (GPO) repeating sequence that is characteristic of all collagen molecules (10, 11). The broad expression pattern of this inhibitory receptor and the possibility that immune cells are able to interact with collagens at many places during their trafficking through the body suggest that LAIR-1 may be an important receptor for modulating immune responses. Multiple studies have shown that the ligation of LAIR-1 by mAb or collagens is able to inhibit activation signals (3, 6 -8, 10, 12, 13).Although the function of LAIR-1 in vivo is currently unknown, it is known that the engagement of LAIR-1 with specific mAb is able to inhibit proliferation of human myeloid leukemic cell lines by inducing programmed cell death independent of Fas/Fas ligand interaction (14). Also, the engagement of LAIR-1 expressed on acute myeloid leukemia blasts, isolated from peripheral blood or bone marrow from patients, inhibits GM-CSF-induced proliferation leading to apoptosis, possibly by a mechanism involving inhibition of GM-CSF-induced AKT activation (15). Moreover, there is a correlation in patients with high-risk B cell chronic lymphocytic leukemia with the absence of LAIR-1, suggesting that the absence of this receptor may be involved in the proliferation of leukemic cells (16). During the course of HIV infection, LAIR-1 expression is abnormally expressed both in naive B cells (17) and in a unique tissue like memory B cell subset that is expanded in HIV-infected patients (18). Finally, the levels of LAIR-2 have been shown to be elevated in the syno...

Molecular mechanisms of the Krüppel-like family of transcription factors (KLFs) have been studied more in proliferating cells than in post-mitotic cells such as neurons. We recently found that KLFs regulate intrinsic axon growth ability in central nervous system (CNS) neurons including retinal ganglion cells, and hippocampal and cortical neurons. With at least 15 of 17 KLF family members expressed in neurons and at least 5 structurally unique subfamilies, it is important to determine how this complex family functions in neurons to regulate the intricate genetic programs of axon growth and regeneration. By characterizing the molecular mechanisms of the KLF family in the nervous system, including binding partners and gene targets, and comparing them to defined mechanisms defined outside the nervous system, we may better understand how KLFs regulate neurite growth and axon regeneration.