Protein Kinase N1 Level Predicts Acute Kidney Injury in Patients Undergoing Cardiac Surgery: A Prospective Cohort Study.

INTRODUCTION: The objective of this study was to examine the utility of protein kinase N1 (PKN1) as a biomarker of cardiac surgery-associated AKI (CSA-AKI). METHODS: A prospective cohort study of 110 adults undergoing on-pump cardiac surgery was conducted. The associations between post-operative PKN1 and CSA-AKI, AKI severity, need for renal replacement therapy (RRT), duration of AKI, length of ICU stay, and post-operative hospital stay were evaluated. RESULTS: Patients were categorized into three groups according to PKN1 tertiles. The incidence of CSA-AKI in the third tertile was 3.4-fold higher than that in the first. PKN1 was an independent risk factor for CSA-AKI. The discrimination of PKN1 to CSA-AKI assessed by ROC curve indicated that the AUC was 0.70, and the best cutoff was 5.025 ng/mL. This group (>5.025 ng/mL) was more likely to develop CSA-AKI (p < 0.001). The combined AUC of EuroSCORE, aortic cross-clamp time, and PKN1 was 0.82 (p < 0.001). A higher level of PKN1 was related to increased need for RRT, longer duration of AKI, and length of ICU and post-operative hospital stays. CONCLUSIONS: PKN1 could be a potential biomarker for the prediction of CSA-AKI. The combination of PKN1, EuroSCORE, and aortic cross-clamp time was likely to predict the occurrence of CSA-AKI.

Role of PKN1 in Retinal Cell Type Formation.

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1-/- animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1-/- retinae showing more NeuroD2+ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1-/- retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32+ α-RGC in Pkn1-/- retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development.

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake.

OBJECTIVE: Glucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain. METHODS: The effect of PGM2L1 on neuronal post-ischemic viability was assessed by siRNA-mediated knockdown of PGM2L1 in primary mouse neurons. Acute mouse brain slices were used to correlate the reduction in G-1,6-BP upon ischemia to changes in carbon metabolism by 13C6-glucose tracing. A drug affinity responsive target stability assay was used to test if G-1,6-BP interacts with the mitochondrial pyruvate carrier (MPC) subunits in mouse brain protein extracts. Human embryonic kidney cells expressing a MPC bioluminescence resonance energy transfer sensor were used to analyze how PGM2L1 overexpression affects MPC activity. The effect of G-1,6-BP on mitochondrial pyruvate uptake and oxygen consumption rates was analyzed in isolated mouse brain mitochondria. PGM2L1 and a predicted upstream kinase were overexpressed in a human neuroblastoma cell line and G-1,6-BP levels were measured. RESULTS: We found that G-1,6-BP in mouse brain slices was quickly degraded upon ischemia and reperfusion. Knockdown of PGM2L1 in mouse neurons reduced post-ischemic viability, indicating that PGM2L1 plays a neuroprotective role. The reduction in G-1,6-BP upon ischemia was not accompanied by alterations in glycolytic rates but we did see a reduced 13C6-glucose incorporation into citrate, suggesting a potential role in mitochondrial pyruvate uptake or metabolism. Indeed, G-1,6-BP interacted with both MPC subunits and overexpression of PGM2L1 increased MPC activity. G-1,6-BP, at concentrations found in the brain, enhanced mitochondrial pyruvate uptake and pyruvate-induced oxygen consumption rates. Overexpression of a predicted upstream kinase inhibited PGM2L1 activity, showing that besides metabolism, also signaling pathways can regulate G-1,6-BP levels. CONCLUSIONS: We provide evidence that G-1,6-BP positively regulates mitochondrial pyruvate uptake and post-ischemic neuronal viability. These compelling data reveal a novel mechanism by which neurons can couple glycolysis-derived pyruvate to the tricarboxylic acid cycle. This process is sensitive to the glycolytic flux, the cell's energetic state, and upstream signaling cascades, offering many regulatory means to fine-tune this critical metabolic step.

PKN1 Exerts Neurodegenerative Effects in an In Vitro Model of Cerebellar Hypoxic-Ischemic Encephalopathy via Inhibition of AKT/GSK3ß Signaling.

We recently identified protein kinase N1 (PKN1) as a negative gatekeeper of neuronal AKT protein kinase activity during postnatal cerebellar development. The developing cerebellum is specifically vulnerable to hypoxia-ischemia (HI), as it occurs during hypoxic-ischemic encephalopathy, a condition typically caused by oxygen deprivation during or shortly after birth. In that context, activation of the AKT cell survival pathway has emerged as a promising new target for neuroprotective interventions. Here, we investigated the role of PKN1 in an in vitro model of HI, using postnatal cerebellar granule cells (Cgc) derived from Pkn1 wildtype and Pkn1-/- mice. Pkn1-/- Cgc showed significantly higher AKT phosphorylation, resulting in reduced caspase-3 activation and improved survival after HI. Pkn1-/- Cgc also showed enhanced axonal outgrowth on growth-inhibitory glial scar substrates, further pointing towards a protective phenotype of Pkn1 knockout after HI. The specific PKN1 phosphorylation site S374 was functionally relevant for the enhanced axonal outgrowth and AKT interaction. Additionally, PKN1pS374 shows a steep decrease during cerebellar development. In summary, we demonstrate the pathological relevance of the PKN1-AKT interaction in an in vitro HI model and establish the relevant PKN1 phosphorylation sites, contributing important information towards the development of specific PKN1 inhibitors.

Protein kinase N1 promotes proliferation and invasion of liver cancer.

Protein kinase (PK) N1, also called PKC-related protein 1, participates in the proliferation, invasion and metastasis of various malignant tumors. However, the role of PKN1 in liver cancer remains to be elucidated. The present study investigated the expression of PKN1 using immunohistochemistry in surgical specimens from 36 patients and analyzed the correlation with VEGF, microvascular density (MVD), cell proliferation index (Ki67) and clinicopathological parameters. PKN1 was highly expressed in hepatocellular carcinoma (HCC) and was positively correlated with histological grading of HCC, Ki67 expression and MVD. PKN1 expression in moderately and poorly differentiated HCC was significantly higher compared with highly differentiated HCC. Expression of PKN1 was positively correlated with Ki67 and MVD, and Ki67 expression was positively correlated with MVD. The effects of PKN1 on proliferation, invasion and apoptosis of liver cancer cells were detected in vitro. Cell viability, migration and invasion were reduced and the apoptosis rate was significantly improved when PKN1 expression was silenced in liver cancer cells. Thus, PKN1 serves an important role in the development and progression of liver cancer. Inhibition of PKN1 activity may provide a promising therapeutic target for liver cancer.

The PKN1- TRAF1 signaling axis as a potential new target for chronic lymphocytic leukemia.

TRAF1 is a pro-survival adaptor molecule in TNFR superfamily (TNFRSF) signaling. TRAF1 is overexpressed in many B cell cancers including refractory chronic lymphocytic leukemia (CLL). Little has been done to assess the role of TRAF1 in human cancer. Here we show that the protein kinase C related kinase Protein Kinase N1 (PKN1) is required to protect TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling in lymphoma. We show that the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but minimally detected in unstimulated healthy donor B cells. Through a screen of 700 kinase inhibitors, we identified two inhibitors, OTSSP167, and XL-228, that inhibited PKN1 in the nanomolar range and induced dose-dependent loss of TRAF1 in RAJI cells. OTSSP167 or XL-228 treatment of primary patient CLL samples led to a reduction in TRAF1, pNF-κB p65, pS6, pERK, Mcl-1 and Bcl-2 proteins, and induction of activated caspase-3. OTSSP167 synergized with venetoclax in inducing CLL death, correlating with loss of TRAF1, Mcl-1, and Bcl-2. Although correlative, these findings suggest the PKN1-TRAF1 signaling axis as a potential new target for CLL. These findings also suggest the use of the orally available inhibitor OTSSP167 in combination treatment with venetoclax for TRAF1 overexpressing CLL.