Relevance of CYP3A5 Expression on the Clinical Outcome of Patients With Renal Cell Carcinoma

Background/Aim: This study aimed to elucidate the detailed characteristics of CYP3A5 expression and the association between CYP3A5 expression and clinical outcomes in patients with renal cell carcinoma (RCC). Patients and Methods: This study retrospectively enrolled 124 Japanese patients with RCC treated at the Okayama University Hospital. The commonest CYP3A5 gene polymorphism, CYP3A5*3, and expression levels of CYP3A5 mRNA and protein in each tissue were examined. Results: Expression of CYP3A5 mRNA and protein in RCC tissues was significantly down-regulated compared to that in adjacent normal tissues. High level of CYP3A5 mRNA expression significantly extended cancer-specific survival (p=0.004) and overall survival (p=0.002). The CYP3A5 mRNA expression level was identified as a significant independent prognostic factor for both cancer-specific survival and overall survival. Conclusion: CYP3A5 could serve as a potential marker for prognostication and treatment planning for patients with RCC

A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment.

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling

A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis

Activation of BMP-Smad1/5/8 Signaling Promotes Survival of Retinal Ganglion Cells after Damage In Vivo

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-Methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma

Genome-Wide Analysis of Müller Glial Differentiation Reveals a Requirement for Notch Signaling in Postmitotic Cells to Maintain the Glial Fate

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10–14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle

Retina-specific loss of Ikbkap/Elp1 causes mitochondrial dysfunction that leads to selective retinal ganglion cell degeneration in a mouse model of familial dysautonomia

Familial dysautonomia (FD) is an autosomal recessive disorder marked by developmental and progressive neuropathies. It is caused by an intronic point-mutation in the IKBKAP/ELP1 gene, which encodes the inhibitor of κB kinase complex-associated protein (IKAP, also called ELP1), a component of the elongator complex. Owing to variation in tissue-specific splicing, the mutation primarily affects the nervous system. One of the most debilitating hallmarks of FD that affects patients' quality of life is progressive blindness. To determine the pathophysiological mechanisms that are triggered by the absence of IKAP in the retina, we generated retina-specific Ikbkap conditional knockout (CKO) mice using Pax6-Cre, which abolished Ikbkap expression in all cell types of the retina. Although sensory and autonomic neuropathies in FD are known to be developmental in origin, the loss of IKAP in the retina did not affect its development, demonstrating that IKAP is not required for retinal development. The loss of IKAP caused progressive degeneration of retinal ganglion cells (RGCs) by 1 month of age. Mitochondrial membrane integrity was breached in RGCs, and later in other retinal neurons. In Ikbkap CKO retinas, mitochondria were depolarized, and complex I function and ATP were significantly reduced. Although mitochondrial impairment was detected in all Ikbkap-deficient retinal neurons, RGCs were the only cell type to degenerate; the survival of other retinal neurons was unaffected. This retina-specific FD model is a useful in vivo model for testing potential therapeutics for mitigating blindness in FD. Moreover, our data indicate that RGCs and mitochondria are promising targets

Intravitreal injection of NMDA results in death of retinal ganglion cells and amacrine cells by 2 days.

<p><b>A.</b> Images of retinal cross sections (left column) and flatmounts (right 3 columns) are shown. Neurotoxic damage induced by 100 mM NMDA injection caused a reduction in Brn3+ (red) retinal ganglion cells and HuC/D+ amacrine (green) cells compared to untreated (NT) or vehicle injected (PBS) retinas by 2 days after injection. GFAP, a marker for retinal damage, was also upregulated at 2 days. Scale bars: 100 µm. <b>B.</b> Brn3+ cells were counted in random flatmount fields. At 2 days after NMDA injection, there was a significant reduction in Brn3+ cells (276.5±29.6 Brn3+ cells/mm²) compared to NT retinas (2409.5±149.9 Brn3+ cells/mm²). No further reduction in Brn3+ cells was observed at 5d after NMDA injection (273.7±29.6 Brn3+ cells/mm²). Therefore, we collected eyes 2 days after injection for the rest of the study. *p<0.005 with t-test.</p

Injection of BMP4 or inhibitors of BMP along with NMDA potentiates or decreases, respectively, Smad1/5/8 activation without affecting Smad2/3 signaling.

<p>Smad1/5/8 and Smad2/3 activation was detected 2 days after the injection of indicated factors by immunohistochemistry. A lower dose of NMDA (10 mM) sufficiently activates both Smad1/5/8 and Smad2/3 in the inner retina. Injection of BMP4 along with NMDA caused a small increase in pSmad1/5/8 over the NMDA alone. When NMDA was co-injected with DM, Smad1/5/8 phosphorylation was blocked effectively without affecting Smad2/3 activation. Co-injection with an additional BMP signaling inhibitor, noggin (Nog), completely blocked Smad1/5/8 activation, and caused slight reduction in pSmad2/3. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganclion cell layer. Scale bars: 30 µm. Representative images from at least 3 animals per treatment are shown.</p

NMDA damage induces expression of Id1, a known target of BMP-Smad1/5/8 signaling.

<p><b>A.</b> Representative images from at least 3 animals per treatment are shown. Injection of 100 mM NMDA induced Id1 expression (red) in Hes5-GFP+ Müller cells (green). Id1 expression was blocked by coinjection of BMP inhibitors, LDN-193189 (LDN) or dorsomorphin (DM). Scale bar: 50 µm. <b>B.</b> Representative Western blot showing the level of Id1 expression 2 days after injection of the indicated factors. <b>C.</b> Quantification of the Western blots. More than a 2 fold increase in Id1 expression was observed after NMDA damage, and this increase was blocked effectively by LDN or DM. The level of Id1 expression was normalized to beta-actin. *p<0.05 (t-test; n = 3 retinas).</p