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Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival.

Wiggins AK, Wei G, Doxakis E, Wong C, Tang AA, Zang K, Luo EJ, Neve RL, Reichardt LF, Huang EJ - J. Cell Biol. (2004)

Bottom Line: Overexpression of HIPK2 induces apoptosis in cultured sensory neurons.Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion.Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
The Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

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HIPK2 induces neuronal apoptosis. (A–H) Although EGFP has no effect on the survival of cultured trigeminal neurons (A–D), EGFP-HIPK2 induces apoptosis (highlighted by TUNEL stain) (E–H). The distribution of EGFP-HIPK2 varies in neurons, with some showing diffuse localization in the nucleus and others in structures similar to nuclear bodies (E, inset). (I and J) HIPK2-induced apoptosis is dose dependent. More than 50% of neurons die at 72 after the addition of 50 MOI of EGFP-HIPK2 HSV, whereas neurons infected by EGFP at 350 MOI have only slight reduction in survival (I). Neurons expressing EGFP-HIPK2 undergo apoptosis over a period of 3–4 d. By the fourth day, >70% of neurons expressing EGFP-HIPK2 show morphological features consistent with cell death, whereas >80% of neurons expressing EGFP continue to survive (J). Dosage of HSV used in J is 100 MOI for both EGFP and EGFP-HIPK2. All data represent mean ± SEM (n = 3). (K) Compared with wild type or Brn3a+/−, overexpression of HIPK2 induces significantly more apoptosis in Brn3a−/− trigeminal neurons (wild type, n = 4, Brn3a+/−, n = 10, and Brn3a−/−, n = 8, t test, * indicates P < 0.05).
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fig3: HIPK2 induces neuronal apoptosis. (A–H) Although EGFP has no effect on the survival of cultured trigeminal neurons (A–D), EGFP-HIPK2 induces apoptosis (highlighted by TUNEL stain) (E–H). The distribution of EGFP-HIPK2 varies in neurons, with some showing diffuse localization in the nucleus and others in structures similar to nuclear bodies (E, inset). (I and J) HIPK2-induced apoptosis is dose dependent. More than 50% of neurons die at 72 after the addition of 50 MOI of EGFP-HIPK2 HSV, whereas neurons infected by EGFP at 350 MOI have only slight reduction in survival (I). Neurons expressing EGFP-HIPK2 undergo apoptosis over a period of 3–4 d. By the fourth day, >70% of neurons expressing EGFP-HIPK2 show morphological features consistent with cell death, whereas >80% of neurons expressing EGFP continue to survive (J). Dosage of HSV used in J is 100 MOI for both EGFP and EGFP-HIPK2. All data represent mean ± SEM (n = 3). (K) Compared with wild type or Brn3a+/−, overexpression of HIPK2 induces significantly more apoptosis in Brn3a−/− trigeminal neurons (wild type, n = 4, Brn3a+/−, n = 10, and Brn3a−/−, n = 8, t test, * indicates P < 0.05).

Mentions: In previous work, we showed that loss of Brn3a led to a dramatic loss of trigeminal neurons due to increased apoptosis (Huang et al., 1999b). These results lead to the prediction that suppression of Brn3a by HIPK2 should mimic Brn3a loss-of-function phenotype and result in neuronal apoptosis. To test this hypothesis, a gain-of-function approach was used to determine the effect of HIPK2 on the survival of cultured sensory neurons. Although herpes simplex virus (HSV)–mediated expression of EGFP did not affect the survival of sensory neurons (Fig. 3, A–D), expression of EGFP-HIPK2 induced apoptosis, indicated by positive TUNEL staining, in a dose-dependent manner (Fig. 3, E–I). Ectopically expressed EGFP-HIPK2 could be detected either diffusely in the nucleus of trigeminal neurons or in structures resembling nuclear bodies (Fig. 3 E, inset). Neurons expressing EGFP-HIPK2 showed TUNEL-positive staining as early as 24 h after infection (Fig. 3, E–H, and J), by 96 h >80% of these neurons were dead as assessed by morphological criteria (Fig. 3 J). In contrast, expression of EGFP had no effect on survival of infected neurons (Fig. 3, I–J). To determine if the pro-apoptotic function of HIPK2 was solely dependent on its interaction with Brn3a, we examined the effect of HIPK2 expression in sensory neurons of Brn3a−/− mutants. One would predict that if HIPK2 function depends exclusively on its ability to suppress Brn3a, HIPK2-induced apoptosis should be attenuated in Brn3a−/− neurons. Alternatively, if HIPK2 continues to induce apoptosis in these neurons, it is likely that HIPK2 may also activate additional Brn3a-independent cell death mechanisms. Our results indicated that, similar to the data in J, ∼30% of wild-type and Brn3a+/− neurons survived at 72 h after infection with HSV-HIPK2 (Fig. 3 K). However, significantly fewer (∼10%) neurons from Brn3a−/− mutants survived under the same treatment (Fig. 3 K). These results support the notion that HIPK2 and Brn3a antagonize each other and the interaction between these two regulates a delicate balance of gene expression critical for neuronal survival. They also suggest that overexpression of HIPK2 in the absence of Brn3a can lead to significantly more apoptosis, probably due to activation of Brn3a-independent cell death pathway(s) (D'Orazi et al., 2002; Hofmann et al., 2002; Zhang et al., 2003).


Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival.

Wiggins AK, Wei G, Doxakis E, Wong C, Tang AA, Zang K, Luo EJ, Neve RL, Reichardt LF, Huang EJ - J. Cell Biol. (2004)

HIPK2 induces neuronal apoptosis. (A–H) Although EGFP has no effect on the survival of cultured trigeminal neurons (A–D), EGFP-HIPK2 induces apoptosis (highlighted by TUNEL stain) (E–H). The distribution of EGFP-HIPK2 varies in neurons, with some showing diffuse localization in the nucleus and others in structures similar to nuclear bodies (E, inset). (I and J) HIPK2-induced apoptosis is dose dependent. More than 50% of neurons die at 72 after the addition of 50 MOI of EGFP-HIPK2 HSV, whereas neurons infected by EGFP at 350 MOI have only slight reduction in survival (I). Neurons expressing EGFP-HIPK2 undergo apoptosis over a period of 3–4 d. By the fourth day, >70% of neurons expressing EGFP-HIPK2 show morphological features consistent with cell death, whereas >80% of neurons expressing EGFP continue to survive (J). Dosage of HSV used in J is 100 MOI for both EGFP and EGFP-HIPK2. All data represent mean ± SEM (n = 3). (K) Compared with wild type or Brn3a+/−, overexpression of HIPK2 induces significantly more apoptosis in Brn3a−/− trigeminal neurons (wild type, n = 4, Brn3a+/−, n = 10, and Brn3a−/−, n = 8, t test, * indicates P < 0.05).
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fig3: HIPK2 induces neuronal apoptosis. (A–H) Although EGFP has no effect on the survival of cultured trigeminal neurons (A–D), EGFP-HIPK2 induces apoptosis (highlighted by TUNEL stain) (E–H). The distribution of EGFP-HIPK2 varies in neurons, with some showing diffuse localization in the nucleus and others in structures similar to nuclear bodies (E, inset). (I and J) HIPK2-induced apoptosis is dose dependent. More than 50% of neurons die at 72 after the addition of 50 MOI of EGFP-HIPK2 HSV, whereas neurons infected by EGFP at 350 MOI have only slight reduction in survival (I). Neurons expressing EGFP-HIPK2 undergo apoptosis over a period of 3–4 d. By the fourth day, >70% of neurons expressing EGFP-HIPK2 show morphological features consistent with cell death, whereas >80% of neurons expressing EGFP continue to survive (J). Dosage of HSV used in J is 100 MOI for both EGFP and EGFP-HIPK2. All data represent mean ± SEM (n = 3). (K) Compared with wild type or Brn3a+/−, overexpression of HIPK2 induces significantly more apoptosis in Brn3a−/− trigeminal neurons (wild type, n = 4, Brn3a+/−, n = 10, and Brn3a−/−, n = 8, t test, * indicates P < 0.05).
Mentions: In previous work, we showed that loss of Brn3a led to a dramatic loss of trigeminal neurons due to increased apoptosis (Huang et al., 1999b). These results lead to the prediction that suppression of Brn3a by HIPK2 should mimic Brn3a loss-of-function phenotype and result in neuronal apoptosis. To test this hypothesis, a gain-of-function approach was used to determine the effect of HIPK2 on the survival of cultured sensory neurons. Although herpes simplex virus (HSV)–mediated expression of EGFP did not affect the survival of sensory neurons (Fig. 3, A–D), expression of EGFP-HIPK2 induced apoptosis, indicated by positive TUNEL staining, in a dose-dependent manner (Fig. 3, E–I). Ectopically expressed EGFP-HIPK2 could be detected either diffusely in the nucleus of trigeminal neurons or in structures resembling nuclear bodies (Fig. 3 E, inset). Neurons expressing EGFP-HIPK2 showed TUNEL-positive staining as early as 24 h after infection (Fig. 3, E–H, and J), by 96 h >80% of these neurons were dead as assessed by morphological criteria (Fig. 3 J). In contrast, expression of EGFP had no effect on survival of infected neurons (Fig. 3, I–J). To determine if the pro-apoptotic function of HIPK2 was solely dependent on its interaction with Brn3a, we examined the effect of HIPK2 expression in sensory neurons of Brn3a−/− mutants. One would predict that if HIPK2 function depends exclusively on its ability to suppress Brn3a, HIPK2-induced apoptosis should be attenuated in Brn3a−/− neurons. Alternatively, if HIPK2 continues to induce apoptosis in these neurons, it is likely that HIPK2 may also activate additional Brn3a-independent cell death mechanisms. Our results indicated that, similar to the data in J, ∼30% of wild-type and Brn3a+/− neurons survived at 72 h after infection with HSV-HIPK2 (Fig. 3 K). However, significantly fewer (∼10%) neurons from Brn3a−/− mutants survived under the same treatment (Fig. 3 K). These results support the notion that HIPK2 and Brn3a antagonize each other and the interaction between these two regulates a delicate balance of gene expression critical for neuronal survival. They also suggest that overexpression of HIPK2 in the absence of Brn3a can lead to significantly more apoptosis, probably due to activation of Brn3a-independent cell death pathway(s) (D'Orazi et al., 2002; Hofmann et al., 2002; Zhang et al., 2003).

Bottom Line: Overexpression of HIPK2 induces apoptosis in cultured sensory neurons.Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion.Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
The Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

Show MeSH
Related in: MedlinePlus