The MLK family mediates c-Jun N-terminal kinase activation in neuronal apoptosis - PubMed (original) (raw)
The MLK family mediates c-Jun N-terminal kinase activation in neuronal apoptosis
Z Xu et al. Mol Cell Biol. 2001 Jul.
Abstract
Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.
Figures
FIG. 1
All five members of the MLK family are expressed in PC12 cells and SCG neurons. Total RNA was purified from naive (N) and neuronally differentiated (D) PC12 cells and rat SCG neurons. RNA was reverse transcribed, and expression levels of MLK1, MLK2, MLK3, DLK, and LZK were evaluated by PCR using specific primers as described in Materials and Methods. Expression in two different passages of PC12 cells was analyzed. GAPDH and cyclophilin served as internal controls. For each primer pair, PCR assays were performed at the same cycle number. All assays were performed at least twice, except that for LZK expression in SCGs, which was done once. Representative results are shown.
FIG. 2
Induction of apoptosis in neuronal PC12 cells by overexpression of MLK family members. (a) Transient expression of wild-type MLK3 but not the kinase-inactive form (d/n) induces cell death. Neuronally differentiated PC12 cells were transfected with empty cloning vector (pCMS.EGFP) or with DNA encoding full-length MLK3 or the kinase-inactive form of MLK3 and assessed for cell viability in the presence of NGF at 2, 3, and 4 days after transfection. The percentage of surviving cells was calculated by normalizing to the numbers of cells transfected with pCMS.EGFP at 2 days after transfection. The data are the means of counts from three wells ± SEM, and similar results were obtained in three additional independent experiments. (b) Overexpression of MLK family members or Rac1 V12 and Cdc42 V12 kills neuronal PC12 cells. Cells were transfected with either empty cloning vector (pCMS.EGFP) or constructs encoding different MLK family members, Rac1 V12, or Cdc42 V12. Three days after transfection, numbers of transfected cells were counted in three replicate wells as described in Materials and Methods. (c) Induction of apoptosis in neuronally differentiated PC12 cells by the expression of different MLK family members and constitutive active forms of Rac1 and Cdc42. Cells were transfected with either empty cloning vector (pCMS.EGFP) or constructs encoding different MLK family members, Rac1 V12, or Cdc42 V12. Three days after transfection, the percentages of apoptotic nuclei were determined by scoring per condition at least 100 Hoechst 33242-stained nuclei of cells expressing EGFP. The values in panels b and c are the means of four independent experiments plus SEM.
FIG. 3
Expression of DLK or MLK3 induces sympathetic neuronal apoptosis. (A) Representative immunofluorescence photomicrographs of cells transfected with control vector, wild-type MLK3, or DLK (data shown here are for DLK; the same results were obtained with MLK3). Sympathetic neurons cultured for 2 days in the presence of NGF were transfected with control vector (pCMS.EGFP) (a to c) or pCMS.EGFP-DLK (d to f). Forty-eight hours later, nuclei of the living cells were stained with Hoechst 33242 as described in Materials and Methods. Normal nuclei are stained blue (c), while nuclei of cells undergoing apoptosis appear light blue (e) because of the intensified signal due to shrinking or condensation of nuclei. Magnifications, ×200 (a) and ×600 (b to f). (B) Sympathetic neurons (prepared as for panel A) were transfected with control vector (pCMS.EGFP) or DNA encoding wild-type or dominant-negative (d/n) forms of DLK. At 24, 48, and 72 h later, numbers of cells with EGFP signal in the same well were counted and the percentages of surviving cells were assessed as described in Materials and Methods. The values are the means for three wells ± SEM, and similar results were obtained in two additional independent experiments. (C) Sympathetic neurons were prepared and transfected as for panel B. Twenty-four hours later, living cells were stained as in panel A, and the proportions of apoptotic nuclei in transfected cells were assessed immediately and 24 h later. The results are the means of three independent experiments plus SEM.
FIG. 3
Expression of DLK or MLK3 induces sympathetic neuronal apoptosis. (A) Representative immunofluorescence photomicrographs of cells transfected with control vector, wild-type MLK3, or DLK (data shown here are for DLK; the same results were obtained with MLK3). Sympathetic neurons cultured for 2 days in the presence of NGF were transfected with control vector (pCMS.EGFP) (a to c) or pCMS.EGFP-DLK (d to f). Forty-eight hours later, nuclei of the living cells were stained with Hoechst 33242 as described in Materials and Methods. Normal nuclei are stained blue (c), while nuclei of cells undergoing apoptosis appear light blue (e) because of the intensified signal due to shrinking or condensation of nuclei. Magnifications, ×200 (a) and ×600 (b to f). (B) Sympathetic neurons (prepared as for panel A) were transfected with control vector (pCMS.EGFP) or DNA encoding wild-type or dominant-negative (d/n) forms of DLK. At 24, 48, and 72 h later, numbers of cells with EGFP signal in the same well were counted and the percentages of surviving cells were assessed as described in Materials and Methods. The values are the means for three wells ± SEM, and similar results were obtained in two additional independent experiments. (C) Sympathetic neurons were prepared and transfected as for panel B. Twenty-four hours later, living cells were stained as in panel A, and the proportions of apoptotic nuclei in transfected cells were assessed immediately and 24 h later. The results are the means of three independent experiments plus SEM.
FIG. 4
The MLK family is upstream of MKK4 and -7 and c-Jun in the apoptotic pathway. (a) A dominant-negative form of c-Jun suppresses death induced by overexpression of Rac1 V12 and Cdc42 V12 as well as by the MLK family. pCMS.EGFP as well as DNA-encoding MLK family members, Rac1 V12, and Cdc42 V12 (all in the pCMS.EGFP vector) were cotransfected with either pCMV vector as a control or the dominant-negative (d/n) form of c-Jun (pCMV-TAM67 c-Jun) as described in Materials and Methods. Three days after transfection, cell numbers were determined as described in Materials and Methods. Cell numbers in cultures transfected with pCMS.EGFP-pCDNA3 and pCMS.EGFP-pCMV were defined as 100% survival, and cell numbers in the other transfected cultures were normalized accordingly. (b) Dominant-negative (d/n) forms of MKK4 and MKK7 suppress death induced by MLK expression. pCMS.EGFP and constructs encoding different MLK family members were cotransfected with either pCDNA3 vector or dominant-negative MKK4 and/or dominant-negative MKK7 (both in pCDNA3) as described in Materials and Methods. In those cotransfected with both dominant-negative MKK4 and dominant-negative MKK7, the amounts of dominant-negative MKK4 and dominant-negative MKK7 DNA were equal and the total amounts of both together (1.25 μg/well) were equal to five times the amount of DNA (0.25 μg/well) with which they were cotransfected. Three days after transfection, cell numbers were counted as described in Materials and Methods. Numbers of cells transfected with pCMS.EGFP-pCDNA3 were defined as 100%, and survival in cultures with other transfections were normalized to these values. The values are the means from three independent experiments plus SEM.
FIG. 5
Overexpression of MLK family members induces cytochrome c release. MLK3 and DLK were transfected into neuronally differentiated PC12 cells (data shown here are for DLK; the same results were obtained with MLK3). Thirty hours later, cells were fixed and immunostained with anti-cytochrome c as described in Materials and Methods. All panels show the same field; arrows point to a cell that has an EGFP signal. (A) Cell transfected with pCMS.EGFP-DLK which shows the EGFP signal. (B) Immunostaining with antibodies against cytochrome c. (C) Cells stained with Hoechst dye 33342. (D) Phase-contrast photomicrograph. Magnification, ×400.
FIG. 6
Death induced by MLK family expression can be suppressed by the general caspase inhibitor BAF. pCMS.EGFP as well as constructs encoding MLK family members MLK1, -2, and -3 and DLK were transfected into neuronal PC12 cells as described in Materials and Methods, and half of the cultures were pretreated and maintained with 25 μM BAF. Three days after transfection, cell numbers were determined as described in Materials and Methods. Numbers of cells transfected with pCMS.EGFP or pCMS.EGFP/BAF were each defined as 100%, and the other transfections were normalized to them. The values are the means of three independent experiments plus SEM.
FIG. 7
Relationships between MLKs, Rac1/Cdc42, and ASK1. (a) Death induced by MLK overexpression can be suppressed significantly by dominant-negative (d/n) forms of other family members and partially by the dominant-negative form of ASK1. pCMS.EGFP or DNA encoding MLK family members in the pCMS.EGFP vector were cotransfected with either pCDNA3 or dominant-negative MLK3 or dominant-negative ASK1 constructs in the pCDNA3 vector. MLK3 was also cotransfected with dominant-negative DLK as described in Materials and Methods. Three days after transfection, the proportions of apoptotic nuclei were determined as described in Materials and Methods. The values are the means of three independent experiments plus SEM. (b) Death induced by Rac1 V12 and Cdc42 V12 can be suppressed by dominant-negative (d/n) forms of MLKs as well as by dominant-negative ASK1. pCMS.EGFP and Rac1 V12 and Cdc42 V12 DNAs in the pCMS.EGFP vector were cotransfected with either the pCDNA3 vector or dominant-negative MLK3, dominant-negative DLK, or dominant-negative ASK1 constructs (each in the latter vector) as described in Materials and Methods. Three days after transfection, the proportions of apoptotic nuclei were determined as described in Materials and Methods. Because the results from cotransfection with pCMS.EGFP-pCDNA3 and pCMS.EGFP cotransfected with dominant-negative MLK3, dominant-negative DLK, or dominant-negative ASK1 DNA were not apparently different from each other, only results obtained with pCMS.EGFP-pCDNA3 are shown. The values are the means of three independent experiments plus SEM.
FIG. 8
(a) Dominant-negative (d/n) forms of MLK3 and DLK protect neuronal PC12 cells from NGF deprivation-induced apoptosis. pCMS.EGFP or DNA encoding dominant-negative forms of MLK3 and DLK in the pCMS.EGFP vector were transfected into neuronal PC12 cells. Two days after transfection, NGF deprivation was performed as described in Materials and Methods. NGF was added back to half of the cultures. Surviving cells were counted 24 and 48 h later. Values from wells with NGF were used as controls for each transfection and defined as 100% survival, and values from cultures without NGF were normalized accordingly. (b) Dominant-negative (d/n) forms of MLK3 and DLK protect rat sympathetic neurons from NGF deprivation-induced cell death. pCMS.EGFP alone or containing DNA encoding dominant-negative MLK3 or DLK was transfected into rat sympathetic neurons, and NGF deprivation was performed as described in Materials and Methods. NGF was added back to half the cultures. Numbers of transfected cells were determined 24 and 48 h after NGF deprivation as described in Materials and Methods. Numbers from cultures with NGF were used as controls for each transfection and defined as 100% survival, and values from NGF deprivation cultures were normalized to them. Values are the means from three replicate cultures plus SEM. Similar results were obtained in two additional independent experiments.
FIG. 9
Elevation of DLK levels and phosphorylation in response to NGF deprivation and UV treatment. (A) DLK response to NGF deprivation (−NGF). PC12 cells were treated with NGF for 7 days, washed with serum-free medium without NGF, replated, and collected 0 (control [Con]), 4, 6, and 12 h later. Cell extracts were prepared and analyzed by Western immunoblotting as described in Materials and Methods. DLK antiserum recognized several species at approximately 95 kDa. This recognition was blocked by the peptide used for preparation of the antiserum, and the fastest-migrating of the recognized species comigrated with the kinase-inactive form of DLK overexpressed in CHO cells (data not shown). The membranes in this and other panels in this figure were stripped and reprobed with ERK1 antiserum. (B) DLK response to UV irradiation. Neuronally differentiated PC12 cells were either left untreated (Con) or exposed to UV light (650 J/m2). Four hours later, cell extracts were prepared and subjected to Western immunoblotting with antisera against DLK and ERK1. (C) Multiple electrophoretic forms of DLK are generated by phosphorylation. Extracts of neuronally differentiated PC12 cells exposed to UV light or deprived of NGF (−NGF) as described above were treated with or without phosphatase (PPase) as described in Materials and Methods. The extracts were then subjected to Western immunoblotting with antisera against DLK and ERK1 as probes.
FIG. 10
Death induced by MLK family members is suppressed by the JNK pathway inhibitor CEP-1347 (200 nM). pCMS.EGFP vector and DNA encoding MLK family members, Rac1 V12, Cdc42 V12, and ΔN ASK1 in pCMS.EGFP were transfected into neuronal PC12 cells. Percentages of apoptotic nuclei were determined as for Fig. 6. The values are means of three independent experiments plus SEM.
FIG. 11
Apoptotic death induced by MLK family members is suppressed by myr-AKT. pCMS.EGFP and DNA encoding MLK family members in pCMS.EGFP were cotransfected with either pCMV6 (control) vector or pCMV6-MyrAKT (MyrAKT) as described in Materials and Methods. Three days after transfection, surviving transfected cells were counted. Cell numbers for pCMS.EGFP-pCMV6 and pCMS.EGFP-pCMV6-MyrAKT were defined as 100% survival, and the cell numbers for other transfections were normalized accordingly. The values are the means from three independent experiments plus SEM.
FIG. 12
Cell death induced by overexpression of ΔN ASK1 is partially suppressed by dominant-negative (d/n) forms of MLK3 and DLK. pCMS.EGFP (GFP) and pCMS.EGFP-ΔNASK1 (ΔN ASK1) were cotransfected with either pCDNA3 vector or constructs encoding dominant-negative MLK3 or dominant-negative DLK (in the pCDNA3 vector) as described in Materials and Methods. Three days after transfection, the proportions of apoptotic nuclei were assessed as described in Materials and Methods. Because the results for pCMS.EGFP-pCDNA3 and pCMS.EGFP cotransfected with dominant-negative MLK3 or dominant-negative DLK were not apparently different from one other, only the results for pCMS.EGFP-pCDNA3 are shown. The values are the means from three independent experiments plus SEM.
FIG. 13
Scheme for the MLK-dependent pathway for apoptotic neuronal death. NGF deprivation of neuronal cells induces the activation of Cdc42 and Rac1, which in turn activate the MLK family members, very likely with the mediation of a scaffold protein(s). Activated MLKs phosphorylate and activate MKK4 and -7 (and possibly other MKK family members) which, in turn, leads to the phosphorylation and activation of JNK and c-Jun subsequently. The activated JNK pathway induces the release of cytochrome c, which activates the caspase cascade and thus leads to cell death. CEP-1347 protects both NGF deprivation and expression of MLK-induced death. The protection is presumably due to its inhibition of the activity of MLK family.
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