Neuron-specific SUMO knockdown suppresses global gene expression response and worsens functional outcome after transient forebrain ischemia in mice.

Small ubiquitin-like modifier (SUMO) conjugation (SUMOylation) plays key roles in neurologic function in health and disease. Neuronal SUMOylation is essential for emotionality and cognition, and this pathway is dramatically activated in post-ischemic neurons, a neuroprotective response to ischemia. It is also known from cell culture studies that SUMOylation modulates gene expression. However, it remains unknown how SUMOylation regulates neuronal gene expression in vivo, in the physiologic state and after ischemia, and modulates post-ischemic recovery of neurologic function. To address these important questions, we used a SUMO1-3 knockdown (SUMO-KD) mouse in which a Thy-1 promoter drives expression of 3 distinct microRNAs against SUMO1-3 to silence SUMO expression specifically in neurons. Wild-type and SUMO-KD mice were subjected to transient forebrain ischemia. Microarray analysis was performed in hippocampal CA1 samples, and neurologic function was evaluated. SUMOylation had opposite effects on neuronal gene expression before and after ischemia. In the physiological state, most genes regulated by SUMOylation were up-regulated in SUMO-KD compared to wild-type mice. Brain ischemia/reperfusion significantly modulated the expression levels of more than 400 genes in wild-type mice, with a majority of those genes upregulated. The extent of this post-ischemic transcriptome change was suppressed in SUMO-KD mice. Moreover, SUMO-KD mice exhibited significantly worse functional outcome. This suggests that suppression of global gene expression response in post-ischemic brain due to SUMO knockdown has a negative effect on post-ischemic neurologic function. Together, our data provide a basis for future studies to mechanistically link SUMOylation to neurologic function in health and disease.

Sumoylation-independent activation of Calcineurin-NFAT-signaling via SUMO2 mediates cardiomyocyte hypertrophy.

The objective of this study was to identify unknown modulators of Calcineurin (Cn)-NFAT signaling. Measurement of NFAT reporter driven luciferase activity was therefore utilized to screen a human cardiac cDNA-library (~107 primary clones) in C2C12 cells through serial dilutions until single clones could be identified. This extensive screening strategy culminated in the identification of SUMO2 as a most efficient Cn-NFAT activator. SUMO2-mediated activation of Cn-NFAT signaling in cardiomyocytes translated into a hypertrophic phenotype. Prohypertrophic effects were also observed in mice expressing SUMO2 in the heart using AAV9 (Adeno-associated virus), complementing the in vitro findings. In addition, increased SUMO2-mediated sumoylation in human cardiomyopathy patients and in mouse models of cardiomyopathy were observed. To decipher the underlying mechanism, we generated a sumoylation-deficient SUMO2 mutant (ΔGG). Surprisingly, ΔGG replicated Cn-NFAT-activation and the prohypertrophic effects of native SUMO2, both in vitro and in vivo, suggesting a sumoylation-independent mechanism. Finally, we discerned a direct interaction between SUMO2 and CnA, which promotes CnA nuclear localization. In conclusion, we identified SUMO2 as a novel activator of Cn-NFAT signaling in cardiomyocytes. In broader terms, these findings reveal an unexpected role for SUMO2 in cardiac hypertrophy and cardiomyopathy, which may open the possibility for therapeutic manipulation of this pathway.

Substantially reduced expression of PIAS1 is associated with colon cancer development.

PURPOSE: Protein inhibitors of activated STATs (PIAS) regulate the interferon-gamma (IFN-gamma) signaling pathway, which has numerous effects on tumor development and tumor cell biology. PIAS's also regulate STAT family members not directly involved in IFN-gamma signaling. This project was designed to assess PIAS1 expression in colon cancer. METHODS: To determine whether PIAS1, one of the PIAS family members, or IFN-gamma signaling pathway components could be used to stratify colon tumors, we stained tissue microarrays for PIAS1, interferon regulatory factor-1 (IRF-1) and STAT1alpha. RESULTS: PIAS1 staining of the colon cancer tissue microarrays indicated a strong correlation of normal colon cells, and adenomas, with high expression of both PIAS1 and IRF-1. CONCLUSION: The PIAS1 results in particular may represent a basis for new approaches for efficiently distinguishing adenomas from colon cancer.

Stat1 and SUMO modification.

Many proteins are known to undergo small ubiquitin-related modifier (SUMO) modification by an E1-, E2-, and E3-dependent ligation process. Recognition that protein inhibitor of activated signal transducers and activators of transcription (STATs) (PIAS) proteins are SUMO E3 ligases raised the possibility that STATs may also be regulated by SUMO modification. Consistent with this possibility, a SUMO-ylation consensus site (PsiKxE; Psi indicates hydrophobic residue, and x indicates any residue) was identified in Stat1 (ie, (702)IKTE(705)), but not in other STATs. Biochemical analysis confirmed that Stat1 K(703) could be SUMO modified in vitro. Mutation of this critical lysine (ie, Stat1(K703R)) yielded a protein that, when expressed in Stat1(-/-) mouse embryonic fibroblasts (MEFs), exhibited enhanced DNA binding and nuclear retention. This was associated with modest changes in transcriptional and antiviral activity. However, mutation of the second critical residue in the SUMO consensus site, E(705) (ie, Stat1(E705A)), yielded a protein with wild-type DNA binding, nuclear retention, and transcriptional and antiviral activity. Similar observations were made when these mutants were expressed in primary Stat1(-/-) macrophages. These observations suggest that although Stat1 can uniquely be SUMO-ylated in vitro, this modification is unlikely to play an important role in regulating Stat1 activity in vivo.