Schermer, Bernhard / Habbig, Sandra / Liebau, Max C - C 9

The dynamic regulation of primary cilia in renal injury repair

Renal tubular epithelial cells have an enormous potential for self repair after acute injury. Here, resorption of primary cilia is a key event to allow re-entry of resident renal tubular epithelial cells into mitosis. We aim to elucidate the biology and function of primary cilia in the kidney and the molecular details of ciliogenesis and ciliary resorption during epithelial maintenance and repair.

Introduction

Acute kidney injury (AKI) is a sudden loss of renal function or a sudden onset of renal damage associated with substantial morbidity and mortality resulting in a high socioeconomic burden. Multiple diseases can cause AKI and acute tubular necrosis constitutes one of the most common and most important histological patterns in AKI. Strikingly, the kidneys display a remarkable potential for repair even after severe tubular injury, which is considered to be the result of a re-entry of resident renal tubular epithelial cells into mitosis.

Yet, the molecular cues that control this potential are unclear. Almost all tubular renal epithelial cells carry a primary cilium at their apical surface, a sensory organelle that projects in an antenna-like fashion into the lumen of the tubule. Cilia are critical regulators of classical signaling pathways including Shh, Wnt and Hippo. They emerge from the basal body, which is a modified centriole anchoring the cilium at the apical surface. In addition, this centriole forms part of the microtubule (MT) organizing center (MTOC) at the spindle pole in dividing cells. Thus, the disassembly of cilia can be regarded as a checkpoint cells have to overcome prior to mitotic re-entry.

Key results of the last 3 years from our group include (1) the identification of a novel mechanism differentially regulating the Hippo downstream effectors YAP and TAZ in acute kidney injury, (2) the investigation of molecular details of how the cilia-associated protein Jade1 affects cell cycle progression in renal epithelial cells, and (3) novel insights into the composition of the ciliary membrane-associated proteome. With a newly developed technique we are currently investigating the molecular details underlying the resorption of ciliary membranes.

Regulation of Hippo effectors through RO

YAP and TAZ are mammalian orthologs of drosophila Yorkie. They act as downstream effectors of Hippo signaling and are important regulators of proliferation, tissue regeneration, and apoptosis. Interestingly, we found differences between YAP and TAZ in response to oxidative stress and could demonstrate that only TAZ but not YAP undergoes reversible s-glutathionylation in response to ROS. Moreover, we observed basal TAZ s-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion (I/R) injury in vivo (see Fig. 1). This novel reversible mechanism by which ROS modulates TAZ activity suggests TAZ to be a redox sensor of the hippo pathway contributing to renal repair mechanisms.

Jade1 phosphorylation and cell cycle regulation

Recently, we identified the PHD zinc finger protein Jade1 as a novel ciliary protein involved in the regulation of Wnt signaling. Following up on this initial work we were able to describe the mechanisms regulating the localization and activity of Jade1 which involve both phosphorylation by casein kinase 1 and by the mitotic kinase Plk1. Ultimately, our data suggest that CK1α phosphorylation of Jade 1S might serve as a molecular switch, turning off chromatin remodelling functions of Jade-1S and allowing timely cell cycle progression. Fig. 2 summarizes the results of our initial two studies.

The ciliary membrane-associated proteome

To identify proteins of the ciliary membrane we targeted the biotinylating engineered enzyme APEX specifically to the plasma membrane of primary cilia. Fig. 3 demonstrates the targeting with STED imaging. This technique allowed us to efficiently pull down components of the ciliary membrane proteome that could be identified by mass spectrometry. We are currently investigating the role of several pathways and key player that we identified with this approach. Moreover, we are analyzing the changes of this proteome during ciliary disassembly and after different perturbations within the cells.

Perspectives

Currently, we are using Crispr/Cas9 mediated genome engineering to generate several cell and mouse models to better understand the in vivo role of the mechanisms described.  With cmAPEX we generated a powerful tool to identify structural components affected by ciliary dynamics throughout cell cycle. Our data serves as the base for functional projects on the molecular mechanisms to gain a better understanding of cilia dynamics in tubular damage, e.g. in AKI.

Selected publications

Gandhirajan, R.K., Jain, M., Walla, B., Johnsen, M., Bartram, M.P., Huynh Anh, M., Rinschen, M.M., Benzing, T., and Schermer, B. (2016). Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ). J Biol Chem. 291(22): p. 11596-607.

Borgal, L., Rinschen, M.M., Dafinger, C., Liebrecht, V.I., Abken, H., Benzing, T., and Schermer, B. (2016). Jade-1S phosphorylation induced by CK1alpha contributes to cell cycle progression. Cell Cycle. 15(8): p. 1034-45.

Johnsen, M., Spath, M.R., Denzel, M.S., Gobel, H., Kubacki, T., Hoyer, K.J., Hinze, Y., Benzing, T., Schermer, B., Antebi, A., Burst, V., and Muller, R.U. (2016). Oral Supplementation of Glucosamine Fails to Alleviate Acute Kidney Injury in Renal Ischemia-Reperfusion Damage. PLoS One. 11(8): p. e0161315.
Schermer, B. and Benzing, T. (2016). Endothelial cilia protect against atherosclerosis. EMBO Rep. 17(2): p. 125-6.

Borgal, L., Rinschen, M.M., Dafinger, C., Hoff, S., Reinert, M.J., Lamkemeyer, T., Lienkamp, S.S., Benzing, T., and Schermer, B. (2014). Casein kinase 1 alpha phosphorylates the Wnt regulator Jade-1 and modulates its activity. J Biol Chem. 289(38): 26344-56.

Kohli, P., Bartram, M.P., Habbig, S., Pahmeyer, C., Lamkemeyer, T., Benzing, T., Schermer, B., and Rinschen, M.M. (2014). Label-free quantitative proteomic analysis of the YAP/TAZ interactome. Am J Physiol Cell Physiol. 306(9): C805-18.

Kurschat, C.E., Muller, R.U., Franke, M., Maintz, D., Schermer, B., and Benzing, T. (2014). An approach to cystic kidney diseases: the clinician's view. Nat Rev Nephrol. 10(12): 687-99.


Former Funding Period 01/2014 - 12/2016

Information from this funding period will not be updated anymore. New research related information is available here.


Prof. Dr. med. Bernhard Schermer

Dept. II of Internal Medicine

Prof. Dr. med. Bernhard Schermer

Principal Investigator C 12
Executive Board Member

bernhard.schermer@uk-koeln.de

Publications - Bernhard Schermer

Link to PubMed

Affiliations

CECAD Cologne
sybacol


PD Dr. med. Sandra Habbig

Dept. of Pediatrics and Youth Medicine

PD Dr. med. Sandra Habbig

Co-Principal Investigator C 9

sandra.habbig@uk-koeln.de,

Work +49 221 478 4319

Dept. of Pediatrics and Youth Medicine
Kerpener Str. 62
50937 Cologne

http://kinderklinik.uk-koeln.de/de/unser-team/aerztlicher-dienst/oberaerzte-und-wissenschaftliche-bereichsleitungen

Publications - Sandra Habbig

Link to PubMed


PD Dr. med. Max C Liebau

Dept. of Children and Adolescent Medicine

PD Dr. med. Max Liebau

Co-Principal Investigator C 12

max.liebau@uk-koeln.de

Publications - Max C Liebau

Link to PubMed

Group Members

Lori Borgal (former PostDoc)
Martyna Brütting (technician)
Claudia Dafinger (PostDoc)
Lena Ebert (PhD candidate)
Laura Frech (MD student)
Rajesh Kumar Gandhirajan(former PostDoc)
Sophie Haumann (Postdoc)
Priyanka Kohli (PhD candidate)
Amrei Mandel (MD student)
Lea Münkner (MD student)
Lukas Schlößer (MD student)
Felix Seelemeyer (MD student)
Gisela Slaats (PostDoc)
Benedikt Walla (MD student)

Figure 1

Fig. 1: Increased cytoplasmic and nuclear expression of TAZ and CTGF, but not YAP after ischemia reperfusion in murine kidneys (from: Gandhirajan et al., JBC, 2016).

Figure 2

Fig. 2: Schematic depicting modes of action of Jade-1S relevant to β-catenin ubiquitination. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase for β-catenin in both the nucleus and the cytosol. The ciliary protein NPHP4 stabilizes a non-phosphorylated form of Jade-1 and enhances translocation of Jade-1 to the nucleus, promoting Wnt inhibition. CK1α phosphorylation abrogates the ability of Jade-1 to inhibit canonical Wnt signaling (from Borgal et al., JBC, 2014).

Figure 3

Fig. 3: STED imaging demonstrating the localization of APEX (green) at the ciliary membrane (marker: Arl13b; Kohli et al, unpublished).