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Transforming Growth Factor-β1 Regulates Cdk5 Activity in Primary Sensory Neurons*

Open AccessPublished:March 28, 2012DOI:https://doi.org/10.1074/jbc.M111.329979
      In addition to many important roles for Cdk5 in brain development and synaptic function, we reported previously that Cdk5 regulates inflammatory pain signaling, partly through phosphorylation of transient receptor potential vanilloid 1 (TRPV1), an important Na+/Ca2+ channel expressed in primary nociceptive afferent nerves. Because TGF-β regulates inflammatory processes and its receptor is expressed in TRPV1-positive afferents, we studied the cross-talk between these two pathways in sensory neurons during experimental peripheral inflammation. We demonstrate that TGF-β1 increases transcription and protein levels of the Cdk5 co-activator p35 through ERK1/2, resulting in an increase in Cdk5 activity in rat B104 neuroblastoma cells. Additionally, TGF-β1 enhances the capsaicin-induced Ca2+ influx in cultured primary neurons from dorsal root ganglia (DRG). Importantly, Cdk5 activity was reduced in the trigeminal ganglia and DRG of 14-day-old TGF-β1 knock-out mice, resulting in reduced Cdk5-dependent phosphorylation of TRPV1. The decreased Cdk5 activity is associated with attenuated thermal hyperalgesia in TGF-β1 receptor conditional knock-out mice, where TGF-β signaling is significantly reduced in trigeminal ganglia and DRG. Collectively, our results indicate that active cross-talk between the TGF-β and Cdk5 pathways contributes to inflammatory pain signaling.

      Introduction

      Cyclin-dependent kinase 5 (Cdk5)
      The abbreviations used are: Cdk5
      cyclin-dependent kinase 5
      TRPV1
      transient receptor potential vanilloid 1
      DRG
      dorsal root ganglia
      TG
      trigeminal ganglia
      SNS
      sensory neuron specific.
      is a proline-directed serine/threonine kinase that belongs to the family of cyclin-dependent protein kinases. Cdk5 kinase activity is mainly present in postmitotic neurons where its activators, p35 and p39, are predominantly expressed (for review see Refs.
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      and
      • Dhariwala F.A.
      • Rajadhyaksha M.S.
      An unusual member of the Cdk family, Cdk5.
      ). Mice lacking either Cdk5 (
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.
      ) or both p35 and p39 (
      • Ko J.
      • Humbert S.
      • Bronson R.T.
      • Takahashi S.
      • Kulkarni A.B.
      • Li E.
      • Tsai L.H.
      p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.
      ) exhibit abnormal corticogenesis and perinatal lethality, underlining a crucial role of Cdk5 activity in the developing brain. Moreover, Cdk5 is critical for neuronal survival (
      • Tanaka T.
      • Veeranna
      • Ohshima T.
      • Rajan P.
      • Amin N.D.
      • Cho A.
      • Sreenath T.
      • Pant H.C.
      • Brady R.O.
      • Kulkarni A.B.
      Neuronal cyclin-dependent kinase 5 activity is critical for survival.
      ) and prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3 (
      • Li B.S.
      • Zhang L.
      • Takahashi S.
      • Ma W.
      • Jaffe H.
      • Kulkarni A.B.
      • Pant H.C.
      Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3.
      ). Although earlier studies focused on delineating the molecular roles of Cdk5 in brain development, more recent work has implicated Cdk5 in many other functions in the mature brain such as memory, learning, and cellular processes leading to neurodegeneration (
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      ). We, and others, have reported that Cdk5 activity participates in the regulation of nociceptive signaling (
      • Pareek T.K.
      • Keller J.
      • Kesavapany S.
      • Pant H.C.
      • Iadarola M.J.
      • Brady R.O.
      • Kulkarni A.B.
      Cyclin-dependent kinase 5 activity regulates pain signaling.
      ,
      • Yang Y.R.
      • He Y.
      • Zhang Y.
      • Li Y.
      • Li Y.
      • Han Y.
      • Zhu H.
      • Wang Y.
      Activation of cyclin-dependent kinase 5 (Cdk5) in primary sensory and dorsal horn neurons by peripheral inflammation contributes to heat hyperalgesia.
      ,
      • Pareek T.K.
      • Kulkarni A.B.
      Cdk5, a new player in pain signaling.
      ). An elevated Cdk5 activity associated with the increased expression of Cdk5 and p35 occurs in nociceptive primary afferent neurons during peripheral inflammation (
      • Pareek T.K.
      • Keller J.
      • Kesavapany S.
      • Pant H.C.
      • Iadarola M.J.
      • Brady R.O.
      • Kulkarni A.B.
      Cyclin-dependent kinase 5 activity regulates pain signaling.
      ). Furthermore, we observed that peripheral inflammation increased Cdk5-mediated phosphorylation of transient receptor potential vanilloid 1 (TRPV1), a ligand-gated ion channel critically involved in thermal and inflammatory pain (
      • Pareek T.K.
      • Keller J.
      • Kesavapany S.
      • Agarwal N.
      • Kuner R.
      • Pant H.C.
      • Iadarola M.J.
      • Brady R.O.
      • Kulkarni A.B.
      Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1.
      ). We subsequently demonstrated that tumor necrosis factor-α (TNF-α) regulates Cdk5 activity during pain signaling through transcriptional activation of p35 (
      • Utreras E.
      • Futatsugi A.
      • Rudrabhatla P.
      • Keller J.
      • Iadarola M.J.
      • Pant H.C.
      • Kulkarni A.B.
      Tumor necrosis factor-α regulates cyclin-dependent kinase 5 activity during pain signaling through transcriptional activation of p35.
      ,
      • Utreras E.
      • Futatsugi A.
      • Pareek T.K.
      • Kulkarni A.B.
      Molecular roles of Cdk5 in pain signaling.
      ). Pharmacological modulation of Cdk5 can produce analgesia or anti-hyperalgesia. We observed that resveratrol, a polyphenolic compound with known analgesic activity, can inhibit Cdk5 activity through decreased expression of p35 (
      • Utreras E.
      • Terse A.
      • Keller J.
      • Iadarola M.J.
      • Kulkarni A.B.
      Resveratrol inhibits Cdk5 activity through regulation of p35 expression.
      ). Intraplantar injection of roscovitine, a Cdk5 inhibitor, or intrathecal administration of Cdk5 siRNA blocked the development of hyperalgesia in a complete Freund's adjuvant model of inflammation (
      • Yang Y.R.
      • He Y.
      • Zhang Y.
      • Li Y.
      • Li Y.
      • Han Y.
      • Zhu H.
      • Wang Y.
      Activation of cyclin-dependent kinase 5 (Cdk5) in primary sensory and dorsal horn neurons by peripheral inflammation contributes to heat hyperalgesia.
      ). Additionally, Cdk5-mediated phosphorylation of the δ-opioid receptor impaired receptor function and attenuated anti-nociceptive tolerance for morphine (
      • Xie W.Y.
      • He Y.
      • Yang Y.R.
      • Li Y.F.
      • Kang K.
      • Xing B.M.
      • Wang Y.
      Disruption of Cdk5-associated phosphorylation of residue threonine 161 of the δ-opioid receptor. Impaired receptor function and attenuated morphine antinociceptive tolerance.
      ). These findings suggest that Cdk5 plays an important role in multiple molecular mechanisms involved in pain signaling and modulation.
      Transforming growth factor-β1 (TGF-β1) is an important member of a superfamily of multifunctional growth factors involved in many cellular processes including cell proliferation, differentiation, migration, and apoptosis (
      • Massagué J.
      The transforming growth factor-β family.
      ). Our earlier studies and those of others on the characterization of the Tgf-β1−/− mouse phenotype confirmed TGF-β1 as a key regulator of inflammation (
      • Shull M.M.
      • Ormsby I.
      • Kier A.B.
      • Pawlowski S.
      • Diebold R.J.
      • Yin M.
      • Allen R.
      • Sidman C.
      • Proetzel G.
      • Calvin D.
      Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease.
      ,
      • Kulkarni A.B.
      • Huh C.G.
      • Becker D.
      • Geiser A.
      • Lyght M.
      • Flanders K.C.
      • Roberts A.B.
      • Sporn M.B.
      • Ward J.M.
      • Karlsson S.
      Transforming growth factor β1 null mutation in mice causes excessive inflammatory response and early death.
      ). TGF-β1−/− mice developed a rapid wasting syndrome and died by 3–4 weeks of age. As early as 2 weeks, these mice displayed multifocal inflammation with massive infiltration of lymphocytes and macrophages into several organs, but principally into the heart, lungs, and salivary glands (
      • Kulkarni A.B.
      • Huh C.G.
      • Becker D.
      • Geiser A.
      • Lyght M.
      • Flanders K.C.
      • Roberts A.B.
      • Sporn M.B.
      • Ward J.M.
      • Karlsson S.
      Transforming growth factor β1 null mutation in mice causes excessive inflammatory response and early death.
      ,
      • Kulkarni A.B.
      • Ward J.M.
      • Yaswen L.
      • Mackall C.L.
      • Bauer S.R.
      • Huh C.G.
      • Gress R.E.
      • Karlsson S.
      Transforming growth factor-β1 null mice. An animal model for inflammatory disorders.
      ). Multiple actions of TGF-β1 have been reported in the CNS. TGF-β1 is normally present at low levels in healthy adult CNS cells, but it is rapidly up-regulated following injury and directly induces expression of several injury-related genes (
      • Wyss-Coray T.
      TGF-β pathway as a potential target in neurodegeneration and Alzheimer.
      ). Although TGF-β1 is known to promote survival of neurons, its precise mechanism is still not clear (
      • Brionne T.C.
      • Tesseur I.
      • Masliah E.
      • Wyss-Coray T.
      Loss of TGF-β1 leads to increased neuronal cell death and microgliosis in mouse brain.
      ,
      • Makwana M.
      • Jones L.L.
      • Cuthill D.
      • Heuer H.
      • Bohatschek M.
      • Hristova M.
      • Friedrichsen S.
      • Ormsby I.
      • Bueringer D.
      • Koppius A.
      • Bauer K.
      • Doetschman T.
      • Raivich G.
      Endogenous transforming growth factor β1 suppresses inflammation and promotes survival in adult CNS.
      ). TGF-β1 has also been implicated in the pathology of Alzheimer disease (
      • Tesseur I.
      • Zou K.
      • Esposito L.
      • Bard F.
      • Berber E.
      • Can J.V.
      • Lin A.H.
      • Crews L.
      • Tremblay P.
      • Mathews P.
      • Mucke L.
      • Masliah E.
      • Wyss-Coray T.
      Deficiency in neuronal TGF-β signaling promotes neurodegeneration and Alzheimer pathology.
      ,
      • Grammas P.
      • Ovase R.
      Cerebrovascular transforming growth factor-β contributes to inflammation in the Alzheimer disease brain.
      ) and its overexpression in astrocytes can lead to excessive deposition of extracellular matrix components in the brain resulting in neurological disease (
      • Wyss-Coray T.
      • Feng L.
      • Masliah E.
      • Ruppe M.D.
      • Lee H.S.
      • Toggas S.M.
      • Rockenstein E.M.
      • Mucke L.
      Increased central nervous system production of extracellular matrix components and development of hydrocephalus in transgenic mice overexpressing transforming growth factor-β1.
      ). Recent reports indicate that TGF-β1 may play a role in migraine and neuropathic pain (
      • Ishizaki K.
      • Takeshima T.
      • Fukuhara Y.
      • Araki H.
      • Nakaso K.
      • Kusumi M.
      • Nakashima K.
      Increased plasma transforming growth factor-β1 in migraine.
      ,
      • Echeverry S.
      • Shi X.Q.
      • Haw A.
      • Liu H.
      • Zhang Z.W.
      • Zhang J.
      Transforming growth factor-β1 impairs neuropathic pain through pleiotropic effects.
      ,
      • Tramullas M.
      • Lantero A.
      • Díaz A.
      • Morchón N.
      • Merino D.
      • Villar A.
      • Buscher D.
      • Merino R.
      • Hurlé J.M.
      • Izpisúa-Belmonte J.C.
      • Hurlé M.A.
      BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-β family in pain modulation.
      ). However, the molecular mechanisms underlying its involvement in nociceptive signaling are still far from clear. Therefore, the main purpose of this study was to explore a possible cross-talk between Cdk5 and TGF-β signaling pathways, and the influence of the cross-talk on inflammation-induced nociceptive responses.
      Here we used in vitro and in vivo approaches to study the role of TGF-β1 in the regulation of Cdk5 activity and its involvement in inflammatory pain signaling. We found that TGF-β1 increases Cdk5 activity in B104 neuroblastoma cells and causes increased Cdk5-dependent TRPV1 phosphorylation and capsaicin-induced Ca2+ influx in dorsal root ganglia (DRG) primary cultures. Likewise, a deficiency of TGF-β signaling in TGF-β1−/− mice, or where transforming growth factor-β receptor 1 (Tgfbr1) is conditionally knocked out in TG and DRG, resulted in reduced Cdk5 activity and attenuated thermal hyperalgesia, suggesting an active cross-talk between TGF-β and Cdk5 pathways in sensory afferents during peripheral inflammatory states.

      DISCUSSION

      In this study, we investigated cross-talk between two important signaling pathways involved in inflammation and how this cross-talk could affect nociceptive signaling. Our investigations demonstrate that in rat neuroblastoma B104 cells, TGF-β1 treatment increased Cdk5 and p35 protein levels with a concomitant increase in Cdk5 activity. Notably, we found TGF-β1 treatment increased Cdk5-mediated phosphorylation of TRPV1 and increased capsaicin-induced calcium influx in primary DRG neuronal cultures. We also report here that Cdk5 activity was decreased in TG and DRG of Tgf-β1−/− mice, resulting in decreased Cdk5-mediated phosphorylation of TRPV1 in DRG. We confirmed these findings in Tgfbr1 cKO mice (SNS-Cre; Tgfbr1f/f) and again observed decreased Cdk5 activity in TG and DRG with attenuated thermal hyperalgesia after peripheral inflammation. Thus, our results indicate that an active cross-talk between the TGF-β and Cdk5 pathways directly influences pain signaling.
      We and others have demonstrated that Cdk5 plays an important role in pain signaling (
      • Pareek T.K.
      • Keller J.
      • Kesavapany S.
      • Pant H.C.
      • Iadarola M.J.
      • Brady R.O.
      • Kulkarni A.B.
      Cyclin-dependent kinase 5 activity regulates pain signaling.
      ,
      • Yang Y.R.
      • He Y.
      • Zhang Y.
      • Li Y.
      • Li Y.
      • Han Y.
      • Zhu H.
      • Wang Y.
      Activation of cyclin-dependent kinase 5 (Cdk5) in primary sensory and dorsal horn neurons by peripheral inflammation contributes to heat hyperalgesia.
      ,
      • Pareek T.K.
      • Kulkarni A.B.
      Cdk5, a new player in pain signaling.
      ,
      • Pareek T.K.
      • Keller J.
      • Kesavapany S.
      • Agarwal N.
      • Kuner R.
      • Pant H.C.
      • Iadarola M.J.
      • Brady R.O.
      • Kulkarni A.B.
      Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1.
      ,
      • Utreras E.
      • Futatsugi A.
      • Rudrabhatla P.
      • Keller J.
      • Iadarola M.J.
      • Pant H.C.
      • Kulkarni A.B.
      Tumor necrosis factor-α regulates cyclin-dependent kinase 5 activity during pain signaling through transcriptional activation of p35.
      ,
      • Utreras E.
      • Futatsugi A.
      • Pareek T.K.
      • Kulkarni A.B.
      Molecular roles of Cdk5 in pain signaling.
      ,
      • Utreras E.
      • Terse A.
      • Keller J.
      • Iadarola M.J.
      • Kulkarni A.B.
      Resveratrol inhibits Cdk5 activity through regulation of p35 expression.
      ,
      • Xie W.Y.
      • He Y.
      • Yang Y.R.
      • Li Y.F.
      • Kang K.
      • Xing B.M.
      • Wang Y.
      Disruption of Cdk5-associated phosphorylation of residue threonine 161 of the δ-opioid receptor. Impaired receptor function and attenuated morphine antinociceptive tolerance.
      ). Previously, we discovered that p35 knock-out mice exhibit significantly decreased Cdk5 activity and show delayed responses to acute noxious thermal stimulation as compared with control mice. In contrast, mice overexpressing p35 exhibit elevated levels of Cdk5 activity and are more sensitive to noxious thermal stimuli than controls. Furthermore, carrageenan-induced (
      • Pareek T.K.
      • Keller J.
      • Kesavapany S.
      • Pant H.C.
      • Iadarola M.J.
      • Brady R.O.
      • Kulkarni A.B.
      Cyclin-dependent kinase 5 activity regulates pain signaling.
      ) or complete Freund's adjuvant-induced (
      • Yang Y.R.
      • He Y.
      • Zhang Y.
      • Li Y.
      • Li Y.
      • Han Y.
      • Zhu H.
      • Wang Y.
      Activation of cyclin-dependent kinase 5 (Cdk5) in primary sensory and dorsal horn neurons by peripheral inflammation contributes to heat hyperalgesia.
      ) inflammation of the mouse hind paw caused increased Cdk5 activity in DRG with a concomitant increase in the phosphorylation of TRPV1 at Thr-407 (
      • Pareek T.K.
      • Keller J.
      • Kesavapany S.
      • Agarwal N.
      • Kuner R.
      • Pant H.C.
      • Iadarola M.J.
      • Brady R.O.
      • Kulkarni A.B.
      Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1.
      ). In addition, we determined that TNF-α is a major regulator of p35 expression during inflammation, thereby directly affecting Cdk5 activity (
      • Utreras E.
      • Futatsugi A.
      • Rudrabhatla P.
      • Keller J.
      • Iadarola M.J.
      • Pant H.C.
      • Kulkarni A.B.
      Tumor necrosis factor-α regulates cyclin-dependent kinase 5 activity during pain signaling through transcriptional activation of p35.
      ,
      • Utreras E.
      • Futatsugi A.
      • Pareek T.K.
      • Kulkarni A.B.
      Molecular roles of Cdk5 in pain signaling.
      ). Recent evidence has shown that TGF-β1 is also involved in pain signaling, but the detailed molecular mechanisms are not fully understood (
      • Ishizaki K.
      • Takeshima T.
      • Fukuhara Y.
      • Araki H.
      • Nakaso K.
      • Kusumi M.
      • Nakashima K.
      Increased plasma transforming growth factor-β1 in migraine.
      ,
      • Echeverry S.
      • Shi X.Q.
      • Haw A.
      • Liu H.
      • Zhang Z.W.
      • Zhang J.
      Transforming growth factor-β1 impairs neuropathic pain through pleiotropic effects.
      ,
      • Tramullas M.
      • Lantero A.
      • Díaz A.
      • Morchón N.
      • Merino D.
      • Villar A.
      • Buscher D.
      • Merino R.
      • Hurlé J.M.
      • Izpisúa-Belmonte J.C.
      • Hurlé M.A.
      BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-β family in pain modulation.
      ,
      • Ronaldson P.T.
      • Finch J.D.
      • Demarco K.M.
      • Quigley C.E.
      • Davis T.P.
      Inflammatory pain signals an increase in functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier.
      ,
      • Ronaldson P.T.
      • Demarco K.M.
      • Sanchez-Covarrubias L.
      • Solinsky C.M.
      • Davis T.P.
      Transforming growth factor-β signaling alters substrate permeability and tight junction protein expression at the blood-brain barrier during inflammatory pain.
      ,
      • Bø S.H.
      • Davidsen E.M.
      • Gulbrandsen P.
      • Dietrichs E.
      • Bovim G.
      • Stovner L.J.
      • White L.R.
      Cerebrospinal fluid cytokine levels in migraine, tension-type headache and cervicogenic headache.
      ). In addition to TGF-β1 activation of canonical Smad pathways, recent reports have shown that TGF-β1 also induces other (no canonical) pathways, including the RhoA and mitogen-activated protein kinase (MAPK) cascades, the latter include extracellular signal-regulated kinases, ERK1/2 (
      • Luo J.
      • Miller M.W.
      Transforming growth factor β1-regulated cell proliferation and expression of neural cell adhesion molecule in B104 neuroblastoma cells. Differential effects of ethanol.
      ,
      • Habashi J.P.
      • Doyle J.J.
      • Holm T.M.
      • Aziz H.
      • Schoenhoff F.
      • Bedja D.
      • Chen Y.
      • Modiri A.N.
      • Judge D.P.
      • Dietz H.C.
      Angiotensin II type 2 receptor signaling attenuates aortic aneurysm in mice through ERK antagonism.
      ). The ERK1/2 signaling pathway is a major regulator of Cdk5 activity through control of Egr-1 and p35 expression (
      • Utreras E.
      • Futatsugi A.
      • Rudrabhatla P.
      • Keller J.
      • Iadarola M.J.
      • Pant H.C.
      • Kulkarni A.B.
      Tumor necrosis factor-α regulates cyclin-dependent kinase 5 activity during pain signaling through transcriptional activation of p35.
      ,
      • Harada T.
      • Morooka T.
      • Ogawa S.
      • Nishida E.
      ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1.
      ). For these reasons, we chose to evaluate whether TGF-β1 can directly induce Cdk5 activity through sustained and robust expression of p35, thereby suggesting a novel molecular mechanism.
      Previously, we found that another cytokine, TNF-α, increased Cdk5 activity through activation of the ERK1/2-Egr-1-p35 signaling pathway in PC12 cells (
      • Utreras E.
      • Futatsugi A.
      • Rudrabhatla P.
      • Keller J.
      • Iadarola M.J.
      • Pant H.C.
      • Kulkarni A.B.
      Tumor necrosis factor-α regulates cyclin-dependent kinase 5 activity during pain signaling through transcriptional activation of p35.
      ). However, the PC12 cells only express low levels of Tgfbr2 and do not respond to TGF-β (
      • Lutz M.
      • Krieglstein K.
      • Schmitt S.
      • ten Dijke P.
      • Sebald W.
      • Wizenmann A.
      • Knaus P.
      Nerve growth factor mediates activation of the Smad pathway in PC12 cells.
      ). To evaluate whether TGF-β1 can regulate Cdk5 kinase activity, we used rat B104 neuroblastoma cells as a model of developing neurons that respond to TGF-β1 (
      • Miller M.W.
      • Mooney S.M.
      • Middleton F.A.
      Transforming growth factor β1 and ethanol affect transcription and translation of genes and proteins for cell adhesion molecules in B104 neuroblastoma cells.
      ). TGF-β1 also regulates the ERK1/2 signaling pathway and gene expression in these cells (
      • Luo J.
      • Miller M.W.
      Transforming growth factor β1-regulated cell proliferation and expression of neural cell adhesion molecule in B104 neuroblastoma cells. Differential effects of ethanol.
      ,
      • Miller M.W.
      • Mooney S.M.
      • Middleton F.A.
      Transforming growth factor β1 and ethanol affect transcription and translation of genes and proteins for cell adhesion molecules in B104 neuroblastoma cells.
      ). Our results show that B104 cells express Cdk5 and p35 protein, and also display Cdk5 kinase activity. Most importantly, we report here that TGF-β1 treatment significantly increases p35 promoter activity. In addition, we found that the TGF-β1 induced an increase of phospho-ERK1/2, and increased Egr-1 and p35 mRNA expression levels, with a subsequent increase of p35 protein levels resulting in an increase of Cdk5 kinase activity. We also noticed that Egr-1 mRNA peaks at about 1 h and returned to basal level at 6 h after TGF-β treatment, whereas the p35 protein level remained higher at 24 h following the treatment. This lag could be attributed to complex transcriptional regulation of Egr-1 and its involvement in multiple transduction cascades (
      • Pagel J.I.
      • Deindl E.
      Early growth response 1, a transcription factor in the cross-fire of signal transduction cascades.
      ). However, the functional significance of TGF-β action was reinforced by our observation that the TGF-β1 inhibitor (SB431542) significantly blocked the TGF-β1-mediated increase of Cdk5 kinase activity. Likewise, MEK1/2 inhibitor U0126 alone, or in combination with TGF-β1, significantly decreased Cdk5 kinase activity, suggesting that the ERK1/2 signaling pathway is a key pathway mediating the cross-talk between TGF-β1 and Cdk5 kinase pathways. Together these results show that TGF-β1 directly activates ERK1/2 leading to an increase in Egr-1 expression and subsequent elevation of p35 expression. This results in an increase in Cdk5 kinase activity.
      We also found that Cdk5-mediated phosphorylation of TRPV1 was regulated by TGF-β1 in DRG primary cultures. Treatment with TGF-β1 increased, whereas treatment with SB431542 decreased the phosphorylation of TRPV1 in DRG cultures. This phosphorylation of TRPV1 has a functional consequence, because treatment with TGF-β1 increased the number of DRG neurons responding to capsaicin. In contrast, treatment with SB431542 blocked the TGF-β1 effect, suggesting that the TGF-β1 signaling pathway needs to be active to sensitize DRG neurons. It is likely, however, that TGF-β1 directly regulates TRPV1 through post-translational mechanisms in these cells, possibly via regulation of Cdk5 activity because TRPV1 mRNA levels remain unchanged in DRG cultures treated with TGF-β1. Although some cells appear hyper-responsive after TGF-β1 treatment in our experiment, the largest observed affect was an increase in the overall number of cells responding to capsaicin. This suggests that Cdk5-mediated phosphorylation of TRPV1 could regulate trafficking of TRPV1 to the membrane. But location of the Cdk5 phosphorylation site on the N-terminal region of TRPV1 also suggests it may regulate protein-protein interactions that influence TRPV1 activity. Collectively, these observations point toward a role for TGF-β1 in the development of inflammation-induced hyperalgesia through direct regulation of Cdk5 activity and Cdk5-mediated phosphorylation of TRPV1.
      TGF-β has been implicated in pain signaling but its precise role is not completely clear. For instance, TGF-β family members negatively modulate pain perception, specifically in mouse models for neuropathic pain (
      • Echeverry S.
      • Shi X.Q.
      • Haw A.
      • Liu H.
      • Zhang Z.W.
      • Zhang J.
      Transforming growth factor-β1 impairs neuropathic pain through pleiotropic effects.
      ,
      • Tramullas M.
      • Lantero A.
      • Díaz A.
      • Morchón N.
      • Merino D.
      • Villar A.
      • Buscher D.
      • Merino R.
      • Hurlé J.M.
      • Izpisúa-Belmonte J.C.
      • Hurlé M.A.
      BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-β family in pain modulation.
      ). Furthermore, TGF-β1 treatment attenuated neuropathic pain (
      • Echeverry S.
      • Shi X.Q.
      • Haw A.
      • Liu H.
      • Zhang Z.W.
      • Zhang J.
      Transforming growth factor-β1 impairs neuropathic pain through pleiotropic effects.
      ), and also, TGF-β family members were implicated in the modulation of chronic and acute pain perception through the regulation of genes encoding endogenous opioids (
      • Tramullas M.
      • Lantero A.
      • Díaz A.
      • Morchón N.
      • Merino D.
      • Villar A.
      • Buscher D.
      • Merino R.
      • Hurlé J.M.
      • Izpisúa-Belmonte J.C.
      • Hurlé M.A.
      BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-β family in pain modulation.
      ). Also, protein levels of TGF-β1 are significantly increased in the plasma (
      • Ishizaki K.
      • Takeshima T.
      • Fukuhara Y.
      • Araki H.
      • Nakaso K.
      • Kusumi M.
      • Nakashima K.
      Increased plasma transforming growth factor-β1 in migraine.
      ) and cerebrospinal fluid (
      • Bø S.H.
      • Davidsen E.M.
      • Gulbrandsen P.
      • Dietrichs E.
      • Bovim G.
      • Stovner L.J.
      • White L.R.
      Cerebrospinal fluid cytokine levels in migraine, tension-type headache and cervicogenic headache.
      ) of patients with migraines. To evaluate in vivo the role of TGF-β1 in nociceptive processes and its involvement in the regulation of Cdk5 activity, we removed TG and DRG from the Tgf-β1−/− mice and assessed the p35 expression levels and Cdk5 kinase activity in those tissues. A lack of TGF-β1 resulted in decreased p35 protein levels in TG and DRG, along with a decrease in Cdk5 activity. Most importantly, we found a parallel decrease in Cdk5 activity and Cdk5-mediated phosphorylation of TRPV1 in TG and DRG from Tgf-β1−/− mice. To circumvent systemic inflammation associated with TGF-β1−/− mice, we generated cKO mice that specifically lack Tgfbr1 in a subpopulation of nociceptive primary afferents of the TG and DRG and evaluated whether a blunted TGF-β signaling pathway could affect Cdk5 activity and pain sensation. Interestingly, we found a decrease in Cdk5 kinase activity and an associated attenuation of behavioral response to thermal stimulation following inflammation in Tgfbr1 cKO mice, corroborating our observations in Tgf-β1−/− mice. Taken together, these results identify a novel molecular mechanism based on cross-talk between TGF-β1 and Cdk5 that appears to contribute to the sensitization of nociceptive signaling.
      It was reported earlier that TGF-β1, among other members of this family, could be negatively modulating pain perception (
      • Echeverry S.
      • Shi X.Q.
      • Haw A.
      • Liu H.
      • Zhang Z.W.
      • Zhang J.
      Transforming growth factor-β1 impairs neuropathic pain through pleiotropic effects.
      ,
      • Tramullas M.
      • Lantero A.
      • Díaz A.
      • Morchón N.
      • Merino D.
      • Villar A.
      • Buscher D.
      • Merino R.
      • Hurlé J.M.
      • Izpisúa-Belmonte J.C.
      • Hurlé M.A.
      BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-β family in pain modulation.
      ). In contrast, our results on Tgf-β1−/− mice are consistent with a positive modulation of pain signaling by TGF-β1. It should be noted that the Cdk5 kinase activity was unchanged in the spinal cords of Tgf-β1−/− mice (data not shown), suggesting that TGF-β1 may play dual roles in different types of persistent pain conditions. At the level of the nociceptive primary afferent neuron, TGF-β may negatively modulate sensitivity by a Cdk5-independent mechanism in neuropathic nerve injury models, and modulate it positively by a Cdk5-dependent mechanism in peripheral inflammation (as examined in this report). In support of our proposal that TGF-β1 plays a positive role in inflammation-induced hyperalgesia, activin A, another member of the TGF-β family, is released during peripheral inflammation. This increases the expression of the calcitonin gene-related peptide and pain sensation (
      • Xu P.
      • Van Slambrouck C.
      • Berti-Mattera L.
      • Hall A.K.
      Activin induces tactile allodynia and increases calcitonin gene-related peptide after peripheral inflammation.
      ). In addition, it was found that activin A mediates hyperalgesia through a mechanism involving acute sensitization of TRPV1 (
      • Zhu W.
      • Xu P.
      • Cuascut F.X.
      • Hall A.K.
      • Oxford G.S.
      Activin acutely sensitizes dorsal root ganglion neurons and induces hyperalgesia via PKC-mediated potentiation of transient receptor potential vanilloid I.
      ). Basal responses to Aδ-fiber and C-fiber stimulation were not changed in Tgfbr1 cKO mice, but after the induction of inflammation with carrageenan we found decreased hyperalgesia at 5 h but not at 24 h after injection, suggesting that deletion of Tgfbr1 in DRG produced a temporally discrete phenotype characterized by reduced nociceptive transmission following the onset of inflammation. As the inflammation progresses, multiple parallel processes are engaged that lead to pain sensitization but are independent of TGF-β1-mediated Cdk5 activity in peripheral afferents. In conclusion, we have shown that an active cross-talk exists between TGF-β and Cdk5 in primary sensory neurons and affects nociceptive signaling during inflammation (Fig. 6). Thus, TGF-β activates Smad-dependent and Smad-independent pathways, which could regulate ERK1/2 activity and in turn induce Egr-1 expression. This results in elevated levels of Cdk5 and p35 mRNA and proteins, as well as a concomitant increase in Cdk5 activity leading to an increase in Cdk5-mediated phosphorylation of TRPV1. When DRG neurons are sensitized or activated, the increased intracellular Ca2+ could also regulate calpain activity, which could generate more p25, resulting in a stronger induction of Cdk5 activity. Similarly, a lack of TGF-β1 or Tgfbr1 decreased p35 protein expression, which in turn decreases Cdk5 activity. Subsequently, we found an associated decrease in Cdk5-mediated phosphorylation of TRPV1. Our in vitro finding suggests that Cdk5 phosphorylation increases the amount of active TRPV1 in the membrane of DRG neurons. We believe our findings on the active cross-talk between Cdk5 and TGF-β pathways in primary afferents and the influence of the cross-talk on nociceptive processing will enhance our understanding of the complex nature of inflammatory pain transduction and primary afferent sensitization.
      Figure thumbnail gr6
      FIGURE 6A proposed model of TGF-β1-mediated regulation of Cdk5 kinase activity. TGF-β1 up-regulates Cdk5 activity through canonical Smad-dependent pathway and noncanonical ERK1/2 pathway, which can be blocked by SB431542 or U0126, respectively. Activation of these pathways induces p35 protein expression through induction of Egr-1, resulting in increased Cdk5 kinase activity. In addition, it increases Cdk5-mediated phosphorylation of TRPV1, ultimately leading to increased intracellular Ca2+ influx in a subpopulation of nociceptive primary sensory neurons. Moreover, increased intracellular Ca2+ levels up-regulate calpain activity resulting in the production of p25, which leads to a further increase in Cdk5 kinase activity.

      Acknowledgments

      We thank Bradford Hall and Zhijun Sun for critical reading of the manuscript, Alfredo Molinolo for analysis of stained section of Tgfr1 cKO mice, and Shelagh Johnson for expert editorial assistance.

      References

        • Dhavan R.
        • Tsai L.H.
        A decade of CDK5.
        Nat. Rev. Mol. Cell Biol. 2001; 2: 749-759
        • Dhariwala F.A.
        • Rajadhyaksha M.S.
        An unusual member of the Cdk family, Cdk5.
        Cell. Mol. Neurobiol. 2008; 28: 351-369
        • Ohshima T.
        • Ward J.M.
        • Huh C.G.
        • Longenecker G.
        • Veeranna
        • Pant H.C.
        • Brady R.O.
        • Martin L.J.
        • Kulkarni A.B.
        Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.
        Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 11173-11178
        • Ko J.
        • Humbert S.
        • Bronson R.T.
        • Takahashi S.
        • Kulkarni A.B.
        • Li E.
        • Tsai L.H.
        p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.
        J. Neurosci. 2001; 21: 6758-6771
        • Tanaka T.
        • Veeranna
        • Ohshima T.
        • Rajan P.
        • Amin N.D.
        • Cho A.
        • Sreenath T.
        • Pant H.C.
        • Brady R.O.
        • Kulkarni A.B.
        Neuronal cyclin-dependent kinase 5 activity is critical for survival.
        J. Neurosci. 2001; 21: 550-558
        • Li B.S.
        • Zhang L.
        • Takahashi S.
        • Ma W.
        • Jaffe H.
        • Kulkarni A.B.
        • Pant H.C.
        Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3.
        EMBO J. 2002; 21: 324-333
        • Pareek T.K.
        • Keller J.
        • Kesavapany S.
        • Pant H.C.
        • Iadarola M.J.
        • Brady R.O.
        • Kulkarni A.B.
        Cyclin-dependent kinase 5 activity regulates pain signaling.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 791-796
        • Yang Y.R.
        • He Y.
        • Zhang Y.
        • Li Y.
        • Li Y.
        • Han Y.
        • Zhu H.
        • Wang Y.
        Activation of cyclin-dependent kinase 5 (Cdk5) in primary sensory and dorsal horn neurons by peripheral inflammation contributes to heat hyperalgesia.
        Pain. 2007; 127: 109-120
        • Pareek T.K.
        • Kulkarni A.B.
        Cdk5, a new player in pain signaling.
        Cell Cycle. 2006; 5: 585-588
        • Pareek T.K.
        • Keller J.
        • Kesavapany S.
        • Agarwal N.
        • Kuner R.
        • Pant H.C.
        • Iadarola M.J.
        • Brady R.O.
        • Kulkarni A.B.
        Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 660-665
        • Utreras E.
        • Futatsugi A.
        • Rudrabhatla P.
        • Keller J.
        • Iadarola M.J.
        • Pant H.C.
        • Kulkarni A.B.
        Tumor necrosis factor-α regulates cyclin-dependent kinase 5 activity during pain signaling through transcriptional activation of p35.
        J. Biol. Chem. 2009; 284: 2275-2284
        • Utreras E.
        • Futatsugi A.
        • Pareek T.K.
        • Kulkarni A.B.
        Molecular roles of Cdk5 in pain signaling.
        Drug Discov. Today Ther. Strateg. 2009; 6: 105-111
        • Utreras E.
        • Terse A.
        • Keller J.
        • Iadarola M.J.
        • Kulkarni A.B.
        Resveratrol inhibits Cdk5 activity through regulation of p35 expression.
        Mol. Pain. 2011; 7: 49
        • Xie W.Y.
        • He Y.
        • Yang Y.R.
        • Li Y.F.
        • Kang K.
        • Xing B.M.
        • Wang Y.
        Disruption of Cdk5-associated phosphorylation of residue threonine 161 of the δ-opioid receptor. Impaired receptor function and attenuated morphine antinociceptive tolerance.
        J. Neurosci. 2009; 29: 3551-3564
        • Massagué J.
        The transforming growth factor-β family.
        Annu. Rev. Cell Biol. 1990; 6: 597-641
        • Shull M.M.
        • Ormsby I.
        • Kier A.B.
        • Pawlowski S.
        • Diebold R.J.
        • Yin M.
        • Allen R.
        • Sidman C.
        • Proetzel G.
        • Calvin D.
        Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease.
        Nature. 1992; 359: 693-699
        • Kulkarni A.B.
        • Huh C.G.
        • Becker D.
        • Geiser A.
        • Lyght M.
        • Flanders K.C.
        • Roberts A.B.
        • Sporn M.B.
        • Ward J.M.
        • Karlsson S.
        Transforming growth factor β1 null mutation in mice causes excessive inflammatory response and early death.
        Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 770-774
        • Kulkarni A.B.
        • Ward J.M.
        • Yaswen L.
        • Mackall C.L.
        • Bauer S.R.
        • Huh C.G.
        • Gress R.E.
        • Karlsson S.
        Transforming growth factor-β1 null mice. An animal model for inflammatory disorders.
        Am. J. Pathol. 1995; 146: 264-275
        • Wyss-Coray T.
        TGF-β pathway as a potential target in neurodegeneration and Alzheimer.
        Curr. Alzheimer Res. 2006; 3: 191-195
        • Brionne T.C.
        • Tesseur I.
        • Masliah E.
        • Wyss-Coray T.
        Loss of TGF-β1 leads to increased neuronal cell death and microgliosis in mouse brain.
        Neuron. 2003; 40: 1133-1145
        • Makwana M.
        • Jones L.L.
        • Cuthill D.
        • Heuer H.
        • Bohatschek M.
        • Hristova M.
        • Friedrichsen S.
        • Ormsby I.
        • Bueringer D.
        • Koppius A.
        • Bauer K.
        • Doetschman T.
        • Raivich G.
        Endogenous transforming growth factor β1 suppresses inflammation and promotes survival in adult CNS.
        J. Neurosci. 2007; 27: 11201-11213
        • Tesseur I.
        • Zou K.
        • Esposito L.
        • Bard F.
        • Berber E.
        • Can J.V.
        • Lin A.H.
        • Crews L.
        • Tremblay P.
        • Mathews P.
        • Mucke L.
        • Masliah E.
        • Wyss-Coray T.
        Deficiency in neuronal TGF-β signaling promotes neurodegeneration and Alzheimer pathology.
        J. Clin. Invest. 2006; 116: 3060-3069
        • Grammas P.
        • Ovase R.
        Cerebrovascular transforming growth factor-β contributes to inflammation in the Alzheimer disease brain.
        Am. J. Pathol. 2002; 160: 1583-1587
        • Wyss-Coray T.
        • Feng L.
        • Masliah E.
        • Ruppe M.D.
        • Lee H.S.
        • Toggas S.M.
        • Rockenstein E.M.
        • Mucke L.
        Increased central nervous system production of extracellular matrix components and development of hydrocephalus in transgenic mice overexpressing transforming growth factor-β1.
        Am. J. Pathol. 1995; 147: 53-67
        • Ishizaki K.
        • Takeshima T.
        • Fukuhara Y.
        • Araki H.
        • Nakaso K.
        • Kusumi M.
        • Nakashima K.
        Increased plasma transforming growth factor-β1 in migraine.
        Headache. 2005; 45: 1224-1228
        • Echeverry S.
        • Shi X.Q.
        • Haw A.
        • Liu H.
        • Zhang Z.W.
        • Zhang J.
        Transforming growth factor-β1 impairs neuropathic pain through pleiotropic effects.
        Mol. Pain. 2009; 5: 16
        • Tramullas M.
        • Lantero A.
        • Díaz A.
        • Morchón N.
        • Merino D.
        • Villar A.
        • Buscher D.
        • Merino R.
        • Hurlé J.M.
        • Izpisúa-Belmonte J.C.
        • Hurlé M.A.
        BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-β family in pain modulation.
        J. Neurosci. 2010; 30: 1502-1511
        • Grynkiewicz G.
        • Poenie M.
        • Tsien R.Y.
        A new generation of Ca2+ indicators with greatly improved fluorescence properties.
        J. Biol. Chem. 1985; 260: 3440-3450
        • Kulkarni A.B.
        • Karlsson S.
        Transforming growth factor-β1 knockout mice. A mutation in one cytokine gene causes a dramatic inflammatory disease.
        Am. J. Pathol. 1993; 143: 3-9
        • Larsson J.
        • Goumans M.J.
        • Sjöstrand L.J.
        • van Rooijen M.A.
        • Ward D.
        • Levéen P.
        • Xu X.
        • ten Dijke P.
        • Mummery C.L.
        • Karlsson S.
        Abnormal angiogenesis but intact hematopoietic potential in TGF-β type I receptor-deficient mice.
        EMBO J. 2001; 20: 1663-1673
        • Agarwal N.
        • Offermanns S.
        • Kuner R.
        Conditional gene deletion in primary nociceptive neurons of trigeminal ganglia and dorsal root ganglia.
        Genesis. 2004; 38: 122-129
        • Cuellar J.M.
        • Manering N.A.
        • Klukinov M.
        • Nemenov M.I.
        • Yeomans D.C.
        Thermal nociceptive properties of trigeminal afferent neurons in rats.
        Mol. Pain. 2010; 6: 39
        • Mitchell K.
        • Bates B.D.
        • Keller J.M.
        • Lopez M.
        • Scholl L.
        • Navarro J.
        • Madian N.
        • Haspel G.
        • Nemenov M.I.
        • Iadarola M.J.
        Ablation of rat TRPV1-expressing Aδ/C-fibers with resiniferatoxin. Analysis of withdrawal behaviors, recovery of function and molecular correlates.
        Mol. Pain. 2010; 6: 94
        • Iadarola M.J.
        • Brady L.S.
        • Draisci G.
        • Dubner R.
        Enhancement of dynorphin gene expression in spinal cord following experimental inflammation. Stimulus specificity, behavioral parameters, and opioid receptor binding.
        Pain. 1988; 35: 313-326
        • Fan R.J.
        • Shyu B.C.
        • Hsiao S.
        Analysis of nocifensive behavior induced in rats by CO2 laser pulse stimulation.
        Physiol. Behav. 1995; 57: 1131-1137
        • Takahashi S.
        • Ohshima T.
        • Cho A.
        • Sreenath T.
        • Iadarola M.J.
        • Pant H.C.
        • Kim Y.
        • Nairn A.C.
        • Brady R.O.
        • Greengard P.
        • Kulkarni A.B.
        Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 1737-1742
        • Lutz M.
        • Krieglstein K.
        • Schmitt S.
        • ten Dijke P.
        • Sebald W.
        • Wizenmann A.
        • Knaus P.
        Nerve growth factor mediates activation of the Smad pathway in PC12 cells.
        Eur. J. Biochem. 2004; 271: 920-931
        • Luo J.
        • Miller M.W.
        Transforming growth factor β1-regulated cell proliferation and expression of neural cell adhesion molecule in B104 neuroblastoma cells. Differential effects of ethanol.
        J. Neurochem. 1999; 72: 2286-2293
        • Miller M.W.
        • Mooney S.M.
        • Middleton F.A.
        Transforming growth factor β1 and ethanol affect transcription and translation of genes and proteins for cell adhesion molecules in B104 neuroblastoma cells.
        J. Neurochem. 2006; 97: 1182-1190
        • Bottenstein J.E.
        • Sato G.H.
        Growth of a rat neuroblastoma cell line in serum-free supplemented medium.
        Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 514-517
        • Harada T.
        • Morooka T.
        • Ogawa S.
        • Nishida E.
        ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1.
        Nat. Cell Biol. 2001; 3: 453-459
        • Kulkarni A.B.
        • Thyagarajan T.
        • Letterio J.J.
        Function of cytokines within the TGF-β superfamily as determined from transgenic and gene knockout studies in mice.
        Curr Mol. Med. 2002; 2: 303-327
        • Wahl S.M.
        • Chen W.
        Transforming growth factor-β-induced regulatory T cells referee inflammatory and autoimmune diseases.
        Arthritis Res. Ther. 2005; 7: 62-68
        • Ronaldson P.T.
        • Finch J.D.
        • Demarco K.M.
        • Quigley C.E.
        • Davis T.P.
        Inflammatory pain signals an increase in functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier.
        J. Pharmacol. Exp. Ther. 2011; 336: 827-839
        • Ronaldson P.T.
        • Demarco K.M.
        • Sanchez-Covarrubias L.
        • Solinsky C.M.
        • Davis T.P.
        Transforming growth factor-β signaling alters substrate permeability and tight junction protein expression at the blood-brain barrier during inflammatory pain.
        J. Cereb. Blood Flow Metab. 2009; 29: 1084-1098
        • Bø S.H.
        • Davidsen E.M.
        • Gulbrandsen P.
        • Dietrichs E.
        • Bovim G.
        • Stovner L.J.
        • White L.R.
        Cerebrospinal fluid cytokine levels in migraine, tension-type headache and cervicogenic headache.
        Cephalalgia. 2009; 29: 365-372
        • Habashi J.P.
        • Doyle J.J.
        • Holm T.M.
        • Aziz H.
        • Schoenhoff F.
        • Bedja D.
        • Chen Y.
        • Modiri A.N.
        • Judge D.P.
        • Dietz H.C.
        Angiotensin II type 2 receptor signaling attenuates aortic aneurysm in mice through ERK antagonism.
        Science. 2011; 332: 361-365
        • Pagel J.I.
        • Deindl E.
        Early growth response 1, a transcription factor in the cross-fire of signal transduction cascades.
        Indian J. Biochem. Biophys. 2011; 48: 226-235
        • Xu P.
        • Van Slambrouck C.
        • Berti-Mattera L.
        • Hall A.K.
        Activin induces tactile allodynia and increases calcitonin gene-related peptide after peripheral inflammation.
        J. Neurosci. 2005; 25: 9227-9235
        • Zhu W.
        • Xu P.
        • Cuascut F.X.
        • Hall A.K.
        • Oxford G.S.
        Activin acutely sensitizes dorsal root ganglion neurons and induces hyperalgesia via PKC-mediated potentiation of transient receptor potential vanilloid I.
        J. Neurosci. 2007; 27: 13770-13780

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