Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness.

Polycystin-1 (PC-1) and 2 (PC-2) are the products of the PKD1 and PKD2 genes, which are mutated in Autosomal Dominant Polycystic Kidney Disease (ADPKD). They form a receptor/channel complex that has been suggested to function as a mechanosensor, possibly activated by ciliary bending in the renal tubule, and resulting in calcium influx. This model has recently been challenged, leaving the question as to which mechanical stimuli activate the polycystins still open. Here, we used a SILAC/Mass-Spec approach to identify intracellular binding partners of tagged-endogenous PC-1 whereby we detected a class of interactors mediating regulation of cellular actomyosin contraction. Accordingly, using gain and loss-of-function cellular systems we found that PC-1 negatively regulates cellular contraction and YAP activation in response to extracellular stiffness. Thus, PC-1 enables cells to sense the rigidity of the extracellular milieu and to respond appropriately. Of note, in an orthologous murine model of PKD we found evidence of increased actomyosin contraction, leading to enhanced YAP nuclear translocation and transcriptional activity. Finally, we show that inhibition of ROCK-dependent actomyosin contraction by Fasudil reversed YAP activation and significantly improved disease progression, in line with recent studies. Our data suggest a possible direct role of PC-1 as a mechanosensor of extracellular stiffness.

The N-Terminal Domain of NPHP1 Folds into a Monomeric Left-Handed Antiparallel Three-Stranded Coiled Coil with Anti-apoptotic Function.

Mutations in the NPHP1 gene, coding for human nephrocystin-1 (NPHP1), cause the autosomal recessive disease nephronophthisis, the most common cause of end-stage renal disease in children and adolescents. The function and structure of NPHP1 are still poorly characterized. NPHP1 presents a modular structure well in keeping with its role as an adaptor protein: it harbors an SH3 domain flanked by two glutamic acid-rich regions and a conserved C-terminal nephrocystin homology domain (NHD). Similar to other NPHP protein family members, its N-terminus contains a putative coiled-coil domain (NPHP1CC) that is supposed to play an important role in NPHP1 self-association and/or protein-protein interactions. Structural studies proving its structure and its oligomerization state are still lacking. Here we demonstrate that NPHP1CC is monomeric in solution and unexpectedly folds into an autonomous domain forming a three-stranded antiparallel coiled coil suitable for protein-protein interactions. Notably, we found that the NPHP1CC shares remarkable structural similarities with the three-stranded coiled coil of the BAG domain protein family, which is known to mediate the anti-apoptotic function of these proteins, suggesting a possible similar role for NPHP1CC. In agreement with this hypothesis, we show that in the context of the full-length protein the NPHP1CC is fundamental to regulate resistance to apoptotic stimuli.

mTORC1 Upregulation Leads to Accumulation of the Oncometabolite Fumarate in a Mouse Model of Renal Cell Carcinoma.

Renal cell carcinomas (RCCs) are common cancers diagnosed in more than 350,000 people each year worldwide. Several pathways are de-regulated in RCCs, including mTORC1. However, how mTOR drives tumorigenesis in this context is unknown. The lack of faithful animal models has limited progress in understanding and targeting RCCs. Here, we generated a mouse model harboring the kidney-specific inactivation of Tsc1. These animals develop cysts that evolve into papillae, cystadenomas, and papillary carcinomas. Global profiling confirmed several metabolic derangements previously attributed to mTORC1. Notably, Tsc1 inactivation results in the accumulation of fumarate and in mTOR-dependent downregulation of the TCA cycle enzyme fumarate hydratase (FH). The re-expression of FH in cellular systems lacking Tsc1 partially rescued renal epithelial transformation. Importantly, the mTORC1-FH axis is likely conserved in human RCC specimens. We reveal a role of mTORC1 in renal tumorigenesis, which depends on the oncometabolite fumarate.

Role of the Polycystins in Cell Migration, Polarity, and Tissue Morphogenesis.

Cystic kidney diseases (CKD) is a class of disorders characterized by ciliary dysfunction and, therefore, belonging to the ciliopathies. The prototype CKD is autosomal dominant polycystic kidney disease (ADPKD), whose mutated genes encode for two membrane-bound proteins, polycystin-1 (PC-1) and polycystin-2 (PC-2), of unknown function. Recent studies on CKD-associated genes identified new mechanisms of morphogenesis that are central for establishment and maintenance of proper renal tubular diameter. During embryonic development in the mouse and lower vertebrates a convergent-extension (CE)-like mechanism based on planar cell polarity (PCP) and cellular intercalation is involved in "sculpting" the tubules into a narrow and elongated shape. Once the appropriate diameter is established, further elongation occurs through oriented cell division (OCD). The polycystins (PCs) regulate some of these essential processes. In this review we summarize recent work on the role of PCs in regulating cell migration, the cytoskeleton, and front-rear polarity. These important properties are essential for proper morphogenesis of the renal tubules and the lymphatic vessels. We highlight here several open questions and controversies. Finally, we try to outline some of the next steps required to study these processes and their relevance in physiological and pathological conditions.

P38MAPK-dependent phosphorylation and degradation of SRC-3/AIB1 and RARalpha-mediated transcription.

Nuclear retinoic acid (RA) receptors (RARs) activate gene expression through dynamic interactions with coregulators in coordination with the ligand and phosphorylation processes. Here we show that during RA-dependent activation of the RARalpha isotype, the p160 coactivator pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3 is phosphorylated by p38MAPK. SRC-3 phosphorylation has been correlated to an initial facilitation of RARalpha-target genes activation, via the control of the dynamics of the interactions of the coactivator with RARalpha. Then, phosphorylation inhibits transcription via promoting the degradation of SRC-3. In line with this, inhibition of p38MAPK markedly enhances RARalpha-mediated transcription and RA-dependent induction of cell differentiation. SRC-3 phosphorylation and degradation occur only within the context of RARalpha complexes, suggesting that the RAR isotype defines a phosphorylation code through dictating the accessibility of the coactivator to p38MAPK. We propose a model in which RARalpha transcriptional activity is regulated by SRC-3 through coordinated events that are fine-tuned by RA and p38MAPK.

The AF-1 and AF-2 domains of RAR gamma 2 and RXR alpha cooperate for triggering the transactivation and the degradation of RAR gamma 2/RXR alpha heterodimers.

In eukaryotic cells, liganded RAR gamma 2/RXR alpha heterodimers activate the transcription of retinoic acid (RA) target genes and then are degraded through the ubiquitin-proteasome pathway. In this study, we dissected the role of the RAR gamma 2 and RXR alpha partners as well as of their respective AF-1 and AF-2 domains in the processes of transactivation and degradation. RAR gamma 2 is the "engine" initiating transcription and its own degradation subsequent to ligand binding. Integrity of its AF-2 domain and phosphorylation of its AF-1 domain are required for both the degradation and the transactivation of the receptor. Deletion of the whole AF-1 domain does not impair these processes but shifts the receptor toward other proteolytic pathways through RXR alpha. In contrast, RXR alpha plays only a modulatory role, cooperating with RAR gamma 2 through its AF-2 domain and its phosphorylated AF-1 domain in both the transcription activity and the degradation of the RAR gamma 2/RXR alpha heterodimers. Our results underline that the AF-1 and AF-2 domains of each heterodimer partner cooperate with one other and that this cooperation is relevant for both the transcription and degradation processes.