Molecular mechanisms of the suppression of axon regeneration by KLF transcription factors

Akintomide Apara, Je ff rey L. Goldberg

1 University of Miami Miller School of Medicine, Miami, FL, USA

2 Shiley Eye Center, University of California San Diego, La Jolla, CA, USA

Molecular mechanisms of the suppression of axon regeneration by KLF transcription factors

Akintomide Apara1, Je ff rey L. Goldberg2

1 University of Miami Miller School of Medicine, Miami, FL, USA

2 Shiley Eye Center, University of California San Diego, La Jolla, CA, USA

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 de fi ned mechanisms de fi ned outside the nervous system, we may better understand how KLFs regulate neurite growth and axon regeneration.

optic nerve; regeneration; axon growth; retina; retinal ganglion cells; spinal cord; transcription factors

Funding:We gratefully acknowledge funding from the National Eye Institute (EY022129 to JLG; P30-EY022589 to UCSD), the DOD (W81XWH-12-1-0254 to JLG), and an unrestricted grant from Research to Prevent Blindness, Inc.

Apara A, Goldberg JL. Molecular mechanisms of the suppression of axon regeneration by KLF transcription factors. Neural Regen Res. 2014;9(15)：1418-1421.

Introduction

Why do neurons in the central nervous system (CNS) fail to regenerate their axons after injury? This fundamental question has obvious implications for human neurodegenerative disease and injury (Moore and Goldberg, 2010). In the CNS, embryonic or neonatal neurons can regenerate their axons aer injury, whereas postnatal or adult neurons cannot (Bregman and Goldberger, 1982; Kunkel-Bagden et al., 1992).is is at least partially attributable to the development of an inhibitory CNS environment, in which glial cells such as mature astrocytes and oligodendrocytes express molecules that suppress axon regeneration for example aer injury in the adult mammalian CNS (Waxman, 1980; Foran and Peterson, 1992). After injury, damaged axons are exposed to myelin- and astrocyte-associated lipids and proteins that actively inhibit axon growth and regeneration (Yiu and He, 2006). For example, astrocytes secrete chondroitin sulfate proteoglycans (CSPGs) (Snow et al., 1990; McKeon et al., 1999; Becker and Becker, 2002; Jones et al., 2002, 2003; Tang et al., 2003) and oligodendrocytes express myelin-derived axon growth inhibitors including Nogo, myelin associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). When such molecules are genetically knocked out (Bartsch et al., 1995; Kim et al., 2003; Simonen et al., 2003; Zheng et al., 2003; Su et al., 2008), neutralized through antibody treatments (Caroni and Schwab, 1988; Bregman et al., 1995; Tang et al., 2001), or enzymatically digested (Crespo et al., 2007) in animal models, modest regeneration and improvement in behavioral assays is observed in animal models (Schmandke et al., 2007). Incomplete regeneration in all of these studies suggested the existence of other pathways involved in regenerative failure and motivated the search for signaling pathways intrinsic to the neurons themselves that may limit their regenerative ability.

Intrinsic control of axon regenerative ability and KLFs

A number of signaling molecules have been identi fi ed to act in neurons to limit regenerative ability. Some of these are trivial extensions of the extrinsic, inhibitory environment, such as receptors for inhibitory glial-associated proteins (Ca ff erty and Strittmatter, 2006) or signaling proteins downstream of these, such as rho and rho kinases (Bertrand et al., 2007). Others are signaling pathways involved more generally in cell growth regulation, such as anaphase promoting complex (APC) (Konishi et al., 2004; Lasorella et al., 2006), and phosphatase and tensin homolog (PTEN; (Park et al., 2008)) or neurotrophic factor responsiveness such as suppressor of cytokine signaling-3 (SOCS3; (Smith et al., 2009)),cyclic adenosine 3′,5′-monophosphate (cAMP; (Cai et al., 2001)), and cAMP response element-binding protein (CREB; (Gao et al., 2004)).

Over the past few decades, multiple studies have demonstrated a developmental decrease in the intrinsic ability of CNS axons to rapidly elongate their axons (Saunders et al., 1992; Treherne et al., 1992; Li et al., 1995; MacLaren and Taylor, 1995; Saunders et al., 1995; Chen et al., 1997; Dusart et al., 1997; Blackmore and Letourneau, 2006), which correlates with the developmental loss of regenerative capacityin vivo. For example, embryonic retinal ganglion cells (RGCs) grow their axons ten-fold faster than postnatal RGCs, and this rapid axon growth ability is lost around the time of birth (Goldberg et al., 2002).

What is the molecular basis for this loss? When we analyzed microarray-derived transcriptomes from different ages of RGCs to identify developmentally regulated genes (Wang et al., 2007), we discovered that expression of the transcription factor Krüppel-like factor 4 (KLF4) signi fi cantly decreased neurite outgrowth in hippocampal and cortical neurons, and RGCs (Moore et al., 2009). Furthermore, KLF4 knockout during early development increased neurite growth from RGCsin vitro, and increased axon regenerationin vivoaer optic nerve injury (Moore et al., 2009). Interestingly, KLF4 expression increases postnatally in RGCs, specifically during the period around birth, which is when RGCs lose their intrinsic axon growth ability (Moore et al., 2009). Moreover, another KLF family member, KLF9, also demonstrated a dramatic 250-fold increase in expression aer birth. Overexpression of KLF9 was also shown to result in a signi fi cant decrease in neurite outgrowthin vitro(Moore et al., 2009). In general, these data support a model whereby the increase in KLF expression around birth, long aer all RGCs have become post-mitotic, leads to a loss of regenerative ability of RGCs. Given KLF9’s higher expression levels aer birth relative to KLF4, and because it was closely related to subfamily members KLF13 and KLF16 that also suppressed axon growth, KLF9 became an intriguing target for studying molecular mechanisms governing its activity in RGCs, but little was known about its regulation or gene targets. We have recently found that knocking down expression of KLF9 with shRNA constructs strongly promotes RGC axon regeneration aer optic nerve injury (unpublished data), and can help to identify both PTMs and downstream target genes required for KLF9 to suppress axon growthin vitroand regenerationin vivo.

Protein-protein interactions are an important regulatory mechanism of transcription factors within the CNS

Many proteins have been shown to interact with and regulate transcription factors in the CNS. Participation in these interactions oen involves post-translational modi fi cations (PTMs) of transcription factors such as acetylation, phosphorylation, ubiquitination, and sumoylation (Savare et al., 2005; Ceballos-Chavez et al., 2012; Goldberg and Trakhtenberg, 2012; Brochier et al., 2013). Such PTMs are critical in functional outcomes such as neuronal differentiation, survival and neurite growth (Cai et al., 2001; Hirata et al., 2003; Savare et al., 2005; Raivich and Makwana, 2007; Lai and Johnson, 2008; Raivich, 2008; Juliandi et al., 2010; Ceballos-Chavez et al., 2012; Hasegawa et al., 2012; Seo et al., 2012; Xie et al., 2012; Brochier et al., 2013; Watkins et al., 2013; Welsbie et al., 2013). Within the KLF family, KLF11 has been shown to interact with both histone acetyl and methyltransferases and these interactions have proven to be crucial in its functional role in regulating the dopamine D2 receptor in dopaminergic neurons (Seo et al., 2012). KLF5 has been shown to be activated by p300 and suppressed by SET in its ability to bind DNA and transactivate downstream target genes (Miyamoto et al., 2003).

KLFs are regulated by phosphorylation

Phosphorylation is perhaps the best studied PTM of transcription factors, and numerous kinase families a ff ect neurite growth and regeneration.e MAPKs (ERKs, JNKs, and p38) and DLK families have been well studied in this regard. MAPK activation upon neuronal injury leads to changes in gene expression patterns mediated by phosphorylated TFs such as c-jun, SOX11, ATF2, P311, and STAT3 (Raivich and Makwana, 2007).ese changes have varied e ff ects including induction of heat shock proteins (HSP25/27) which spur the repair of the actin micro fi lament network (Stokoe et al., 1992; Cohen, 1996), promotion of survival by mitochondrial cytochrome c interference preventing caspase activation (Benn et al., 2002; Hirata et al., 2003; Dodge et al., 2006), and induction of programmed cell death (PCD) upon NGF withdrawalviarelease of inflammatory cytokines such as IL-1 and TNF-α (Xia et al., 1995).

Within the KLF family, we have shown that a MAPK family member regulates KLF9 at two critical residues and that this regulation is crucial to its functional role as a neurite outgrowth suppressor in RGCs (unpublished data). This form of regulationviaphosphorylation is not unique to KLF9 among the KLF family. KLF6 has been shown to be regulated by phosphorylation in COS-7 cell lines metabolically labeled with radioactive phosphate, where it acts as a tumor suppressor (Slavin et al., 2004), but the kinase responsible remains unidenti fi ed. Using protein kinase inhibitors, it was demonstrated that KLF5 is activated by phosphorylation in its role as an oncogene and interacts with protein kinase C (PKC) and p38 but not MAPK in both human pancreatic and breast cancer cell lines (Zhang and Teng, 2003; Mori et al., 2009). KLF11 has been shown to be phosphorylated in 2 linker regions by ERK in CHO cell lines using in vitro phosphorylation assays (Ellenrieder et al., 2002; Ellenrieder, 2008). KLF4 is phosphorylated by PKC at T401, an important regulatory residue, in vascular smooth muscle cell differentiationviaSMADs in a TGF-β and p38 dependent cascade (Zhang et al., 2012). KLF9, however, is thus far unique both in its capacity to bind the MAPK family of kinases and known to be regulated by them within neurons. Besides KLF9, only KLF10 and -11 possess the same structural motif necessary for KLF9-MAPK interaction. This might place these KLFs in signaling modules dependent on these speci fi c kinases.e involvement of these kinases in the regulationof KLFs outside the CNS makes them potential players in the intrinsic control of neurite growth both during normal neuronal development and after injury within the CNS. Characterizing the phosphorylation and phospho-regulation of other KLF family members within the CNS is thus an important goal for future study.

Conclusions

Author statements:Goldberg JL is a co-inventor on a patent assigned to the University of Miami regarding KLFs in regeneration.

Author contributions:Apora A and Goldberg JL wrote the paper. Both of these two authors approved the final version of this manuscript.

Con fl icts of interest:The authors declare no conflicts of interest.

Bartsch U, Bandtlow CE, Schnell L, Bartsch S, Spillmann AA, Rubin BP, Hillenbrand R, Montag D, Schwab ME, Schachner M (1995) Lack of evidence that myelin-associated glycoprotein is a major inhibitor of axonal regeneration in the CNS. Neuron 15：1375-1381.

Becker CG, Becker T (2002) Repellent guidance of regenerating optic axons by chondroitin sulfate glycosaminoglycans in zebra fi sh. J Neurosci 22：842-853.

Benn SC, Perrelet D, Kato AC, Scholz J, Decosterd I, Mannion RJ, Bakowska JC, Woolf CJ (2002) Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival. Neuron 36：45-56.

Bertrand J, Di Polo A, McKerracher L (2007) Enhanced survival and regeneration of axotomized retinal neurons by repeated delivery of cell-permeable C3-like Rho antagonists. Neurobiol Dis 25：65-72.

Blackmore M, Letourneau PC (2006) Changes within maturing neurons limit axonal regeneration in the developing spinal cord. J Neurobiol 66：348-360.

Bregman BS, Goldberger ME (1982) Anatomical plasticity and sparing of function after spinal cord damage in neonatal cats. Science 217：553-555.

Bregman BS, Kunkel-Bagden E, Schnell L, Dai HN, Gao D, Schwab ME (1995) Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature 378：498-501.

Brochier C, Dennis G, Rivieccio MA, McLaughlin K, Coppola G, Ratan RR, Langley B (2013) Speci fi c acetylation of p53 by HDAC inhibition prevents DNA damage-induced apoptosis in neurons. J Neurosci 33：8621-8632.

Cafferty WB, Strittmatter SM (2006) The Nogo-Nogo receptor pathway limits a spectrum of adult CNS axonal growth. J Neurosci 26：12242-12250.

Cai D, Qiu J, Cao Z, McAtee M, Bregman BS, Filbin MT (2001) Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate. J Neurosci 21：4731-4739.

Caroni P, Schwab ME (1988) Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter. Neuron 1：85-96.

Ceballos-Chavez M, Rivero S, Garcia-Gutierrez P, Rodriguez-Paredes M, Garcia-Dominguez M, Bhattacharya S, Reyes JC (2012) Control of neuronal differentiation by sumoylation of BRAF35, a subunit of the LSD1-CoREST histone demethylase complex. Proc Natl Acad Sci U S A 109：8085-8090.

Chen DF, Schneider GE, Martinou JC, Tonegawa S (1997) Bcl-2 promotes regeneration of severed axons in mammalian CNS. Nature 385：434-439.

Cohen P (1996) Dissection of protein kinase cascades that mediate cellular response to cytokines and cellular stress. Adv Pharmacol 36：15-27.

Crespo D, Asher RA, Lin R, Rhodes KE, Fawcett JW (2007) How does chondroitinase promote functional recovery in the damaged CNS? Exp Neurol 206：159-171.

Dodge ME, Wang J, Guy C, Rankin S, Rahimtula M, Mearow KM (2006) Stress-induced heat shock protein 27 expression and its role in dorsal root ganglion neuronal survival. Brain Res 1068：34-48.

Dusart I, Airaksinen MS, Sotelo C (1997) Purkinje cell survival and axonal regeneration are age dependent： an in vitro study. J Neurosci 17：3710-3726.

Ellenrieder V (2008) TGFbeta regulated gene expression by Smads and Sp1/KLF-like transcription factors in cancer. Anticancer Res 28：1531-1539.

Ellenrieder V, Zhang JS, Kaczynski J, Urrutia R (2002) Signaling disrupts mSin3A binding to the Mad1-like Sin3-interacting domain of TIEG2, an Sp1-like repressor. EMBO J 21：2451-2460.

Foran DR, Peterson AC (1992) Myelin acquisition in the central nervous system of the mouse revealed by an MBP-Lac Z transgene. J Neurosci 12：4890-4897.

Gao Y, Deng K, Hou J, Bryson JB, Barco A, Nikulina E, Spencer T, Mellado W, Kandel ER, Filbin MT (2004) Activated CREB is suf fi cient to overcome inhibitors in myelin and promote spinal axon regeneration in vivo. Neuron 44：609-621.

Goldberg JL, Trakhtenberg EF (2012) Axon growth and regeneration：part 1. Preface. Int Rev Neurobiol 105：xi-xiii.

Goldberg JL, Klassen MP, Hua Y, Barres BA (2002) Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells. Science 296：1860-1864.

Hasegawa K, Kawahara T, Fujiwara K, Shimpuku M, Sasaki T, Kitamura T, Yoshikawa K (2012) Necdin controls Foxo1 acetylation in hypothalamic arcuate neurons to modulate the thyroid axis. J Neurosci 32：5562-5572.

Hirata K, He J, Hirakawa Y, Liu W, Wang S, Kawabuchi M (2003) HSP27 is markedly induced in Schwann cell columns and associated regenerating axons. Glia 42：1-11.

Jones LL, Yamaguchi Y, Stallcup WB, Tuszynski MH (2002) NG2 is a major chondroitin sulfate proteoglycan produced after spinal cord injury and is expressed by macrophages and oligodendrocyte progenitors. J Neurosci 22：2792-2803.

Jones LL, Margolis RU, Tuszynski MH (2003) The chondroitin sulfate proteoglycans neurocan, brevican, phosphacan, and versican are differentially regulated following spinal cord injury. Exp Neurol 182：399-411.

Juliandi B, Abematsu M, Nakashima K (2010) Epigenetic regulation in neural stem cell differentiation. Dev Growth Differ 52：493-504.

Kim JE, Li S, GrandPre T, Qiu D, Strittmatter SM (2003) Axon regeneration in young adult mice lacking Nogo-A/B. Neuron 38：187-199.

Konishi Y, Stegmuller J, Matsuda T, Bonni S, Bonni A (2004) Cdh1-APC controls axonal growth and patterning in the mammalian brain. Science 303：1026-1030.

Kunkel-Bagden E, Dai HN, Bregman BS (1992) Recovery of function after spinal cord hemisection in newborn and adult rats： differential effects on re fl ex and locomotor function. Exp Neurol 116：40-51.

Lai HC, Johnson JE (2008) Neurogenesis or neuronal specification：phosphorylation strikes again! Neuron 58：3-5.

Lasorella A, Stegmuller J, Guardavaccaro D, Liu G, Carro MS, Rothschild G, de la Torre-Ubieta L, Pagano M, Bonni A, Iavarone A (2006) Degradation of Id2 by the anaphase-promoting complex couples cell cycle exit and axonal growth. Nature 442：471-474.

Li D, Field PM, Raisman G (1995) Failure of axon regeneration in postnatal rat entorhinohippocampal slice coculture is due to maturation of the axon, not that of the pathway or target. Eur J Neurosci 7：1164-1171.

MacLaren RE, Taylor JS (1995) A critical period for axon regrowth through a lesion in the developing mammalian retina. Eur J Neurosci 7：2111-2118.

McKeon RJ, Jurynec MJ, Buck CR (1999) The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar. J Neurosci 19：10778-10788.

Miyamoto S, Suzuki T, Muto S, Aizawa K, Kimura A, Mizuno Y, Nagino T, Imai Y, Adachi N, Horikoshi M, Nagai R (2003) Positive and negative regulation of the cardiovascular transcription factor KLF5 by p300 and the oncogenic regulator SET through interaction and acetylation on the DNA-binding domain. Mol Cell Biol 23：8528-8541.

Moore DL, Goldberg JL (2010) Four steps to optic nerve regeneration. J Neuroophthalmol 30：347-360.

Moore DL, Blackmore MG, Hu Y, Kaestner KH, Bixby JL, Lemmon VP, Goldberg JL (2009) KLF family members regulate intrinsic axon regeneration ability. Science 326：298-301.

Mori A, Moser C, Lang SA, Hackl C, Gottfried E, Kreutz M, Schlitt HJ, Geissler EK, Stoeltzing O (2009) Up-regulation of Kruppel-like factor 5 in pancreatic cancer is promoted by interleukin-1beta signaling and hypoxia-inducible factor-1alpha. Mol Cancer Res 7：1390-1398.

Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, Xu B, Connolly L, Kramvis I, Sahin M, He Z (2008) Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 322：963-966.

Raivich G (2008) c-Jun expression, activation and function in neural cell death, in fl ammation and repair. J Neurochem 107：898-906.

Raivich G, Makwana M (2007) The making of successful axonal regeneration： genes, molecules and signal transduction pathways. Brain Res Rev 53：287-311.

Saunders NR, Balkwill P, Knott G, Habgood MD, Mollgard K, Treherne JM, Nicholls JG (1992) Growth of axons through a lesion in the intact CNS of fetal rat maintained in long-term culture. Proc Biol Sci 250：171-180.

Saunders NR, Deal A, Knott GW, Varga ZM, Nicholls JG (1995) Repair and recovery following spinal cord injury in a neonatal marsupial (Monodelphis domestica). Clin Exp Pharmacol Physiol 22：518-526.

Savare J, Bonneaud N, Girard F (2005) SUMO represses transcriptional activity of the Drosophila SoxNeuro and human Sox3 central nervous system-specific transcription factors. Mol Biol Cell 16：2660-2669.

Schmandke A, Schmandke A, Strittmatter SM (2007) ROCK and Rho：biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist 13：454-469.

Seo S, Lomberk G, Mathison A, Buttar N, Podratz J, Calvo E, Iovanna J, Brimijoin S, Windebank A, Urrutia R (2012) Kruppel-like factor 11 differentially couples to histone acetyltransferase and histone methyltransferase chromatin remodeling pathways to transcriptionally regulate dopamine D2 receptor in neuronal cells. J Biol Chem 287：12723-12735.

Simonen M, Pedersen V, Weinmann O, Schnell L, Buss A, Ledermann B, Christ F, Sansig G, van der Putten H, Schwab ME (2003) Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury. Neuron 38：201-211.

Slavin DA, Koritschoner NP, Prieto CC, Lopez-Diaz FJ, Chatton B, Bocco JL (2004) A new role for the Kruppel-like transcription factor KLF6 as an inhibitor of c-Jun proto-oncoprotein function. Oncogene 23：8196-8205.

Smith PD, Sun F, Park KK, Cai B, Wang C, Kuwako K, Martinez-Carrasco I, Connolly L, He Z (2009) SOCS3 deletion promotes optic nerve regeneration in vivo. Neuron 64：617-623.

Snow DM, Lemmon V, Carrino DA, Caplan AI, Silver J (1990) Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp Neurol 109：111-130.

Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M (1992) Identi fication of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313：307-313.

Su Y, Wang F, Zhao SG, Pan SH, Liu P, Teng Y, Cui H (2008) Axonal regeneration after optic nerve crush in Nogo-A/B/C knockout mice. Mol Vis 14：268-273.

Tang S, Qiu J, Nikulina E, Filbin MT (2001) Soluble myelin-associated glycoprotein released from damaged white matter inhibits axonal regeneration. Mol Cell Neurosci 18：259-269.

Tang X, Davies JE, Davies SJ (2003) Changes in distribution, cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue. J Neurosci Res 71：427-444.

Treherne JM, Woodward SK, Varga ZM, Ritchie JM, Nicholls JG (1992) Restoration of conduction and growth of axons through injured spinal cord of neonatal opossum in culture. Proc Natl Acad Sci U S A 89：431-434.

Wang JT, Kunzevitzky NJ, Dugas JC, Cameron M, Barres BA, Goldberg JL (2007) Disease gene candidates revealed by expression pro fi ling of retinal ganglion cell development. J Neurosci 27：8593-8603.

Watkins TA, Wang B, Huntwork-Rodriguez S, Yang J, Jiang Z, Eastham-Anderson J, Modrusan Z, Kaminker JS, Tessier-Lavigne M, Lewcock JW (2013) DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury. Proc Natl Acad Sci U S A 110：4039-4044.

Waxman SG (1980) Determinants of conduction velocity in myelinated nerve fi bers. Muscle Nerve 3：141-150.

Welsbie DS et al. (2013) Functional genomic screening identi fi es dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc Natl Acad Sci U S A 110：4045-4050.

Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270：1326-1331.

Xie Q, Hao Y, Tao L, Peng S, Rao C, Chen H, You H, Dong MQ, Yuan Z (2012) Lysine methylation of FOXO3 regulates oxidative stress-induced neuronal cell death. EMBO Rep 13：371-377.

Yiu G, He Z (2006) Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 7：617-627.

Zhang XH, Zheng B, Gu C, Fu JR, Wen JK (2012) TGF-beta1 downregulates AT1 receptor expression via PKC-delta-mediated Sp1 dissociation from KLF4 and Smad-mediated PPAR-gamma association with KLF4. Arterioscler Thromb Vasc Biol 32：1015-1023.

Zhang Z, Teng CT (2003) Phosphorylation of Kruppel-like factor 5 (KLF5/IKLF) at the CBP interaction region enhances its transactivation function. Nucleic Acids Res 31：2196-2208.

Zheng B, Ho C, Li S, Keirstead H, Steward O, Tessier-Lavigne M (2003) Lack of enhanced spinal regeneration in Nogo-de fi cient mice. Neuron 38：213-224.

Jeffrey L. Goldberg, M.D., Ph.D., Shiley Eye Center, University of California San Diego, 9415 Campus Point Dr. #0946, La Jolla, CA 92093, USA, jlgoldberg@ucsd.edu.

10.4103/1673-5374.139454

http://www.nrronline.org/

Accepted: 2014-07-22