Mutations in polycystin-1 (PC1) lead to autosomal-dominant polycystic kidney disease (ADPKD), a leading cause of renal failure for which no treatment is available. PC1 is an integral membrane protein, which has been implicated in the regulation of multiple signaling pathways including the JAK/STAT pathway. Here we show that membrane-anchored PC1 activates STAT3 in a JAK2-dependent manner, leading to tyrosine phosphorylation and transcriptional activity. The C-terminal cytoplasmic tail of PC1 can undergo proteolytic cleavage and nuclear translocation. Tail-cleavage abolishes the ability of PC1 to directly activate STAT3 but the cleaved PC1 tail now coactivates STAT3 in a mechanism requiring STAT phosphorylation by cytokines or growth factors. This leads to an exaggerated cytokine response. Hence, PC1 can regulate STAT activity by a dual mechanism. In ADPKD kidneys PC1 tail fragments are overexpressed, including a unique ∼15-kDa fragment (P15). STAT3 is strongly activated in cyst-lining epithelial cells in human ADPKD, and orthologous and nonorthologous polycystic mouse models. STAT3 is also activated in developing, postnatal kidneys but inactivated in adult kidneys. These results indicate that STAT3 signaling is regulated by PC1 and is a driving factor for renal epithelial proliferation during normal renal development and during cyst growth.A utosomal-dominant polycystic kidney disease (ADPKD) is a common life-threatening genetic disease and leading cause of renal failure (1-4). Epithelial-lined cysts develop due to excessive proliferation leading to renal enlargement and destruction of functional renal tissue. No treatment is available to slow disease progression. PKD1 gene mutations cause the majority of cases but the exact function of its gene product, polycystin-1 (PC1), has remained poorly understood. Confusing the issue is the fact that both loss of PC1 as well as PC1 overexpression lead to renal cyst growth in mouse models. Furthermore, in human ADPKD, each clonal cyst within the same patient is thought to exhibit a unique combination of germline and acquired mutations that results in a cyst-by-cyst mosaic of genotypes and resulting variation of expression levels of PC1 harboring various mutations. PC1 has also been implicated in a puzzling variety of intracellular signaling events including JAK-STAT signaling (5, 6) and it has been difficult to elucidate which of these functions may be most important for renal cyst growth in ADPKD.Disruption of primary cilia in kidney epithelial cells leads to proliferation and cyst growth (7). PC1 has been shown to function in ciliary mechanotransduction, and a potential molecular mechanism emerged from the discovery that PC1 undergoes proteolytic cleavage, releasing the C-terminal cytoplasmic tail (∼30 kDa) from the membrane, followed by nuclear translocation (2, 5). PC1 cleavage is triggered upon the cessation of luminal fluid flow (2). The cleaved PC1 tail interacts with the transcription factors STAT6 and P100, enhances STAT6 activity, and STAT6 translocates between ...

Polycystin-1 (PC1) mutations result in proliferative renal cyst growth and progression to renal failure in autosomal dominant polycystic kidney disease (ADPKD). The transcription factor STAT3 (signal transducer and activator of transcription 3) was shown to be activated in cyst-lining cells in ADPKD and PKD mouse models and may drive renal cyst growth, but the mechanisms leading to persistent STAT3 activation are unknown. A proteolytic fragment of PC1 corresponding to the cytoplasmic tail, PC1-p30, is overexpressed in ADPKD. Here, we show that PC1-p30 interacts with the nonreceptor tyrosine kinase Src, resulting in Srcdependent activation of STAT3 by tyrosine phosphorylation. The PC1-p30-mediated activation of Src/ STAT3 was independent of JAK family kinases and insensitive to the STAT3 inhibitor suppressor of cytokine signaling 3. Signaling by the EGF receptor (EGFR) or cAMP amplified the activation of Src/STAT3 by PC1-p30. Expression of PC1-p30 changed the cellular response to cAMP signaling. In the absence of PC1-p30, cAMP dampened EGFR-or IL-6-dependent activation of STAT3; in the presence of PC1-p30, cAMP amplified Src-dependent activation of STAT3. In the polycystic kidney (PCK) rat model, activation of STAT3 in renal cystic cells depended on vasopressin receptor 2 (V2R) signaling, which increased cAMP levels. Genetic inhibition of vasopressin expression or treatment with a pharmacologic V2R inhibitor strongly suppressed STAT3 activation and reduced renal cyst growth. These results suggest that PC1, via its cleaved cytoplasmic tail, integrates signaling inputs from EGFR and cAMP, resulting in Src-dependent activation of STAT3 and a proliferative response.

Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disease caused by mutations in the gene coding for polycystin-1 (PC1). PC1 can regulate STAT transcription factors by a novel, dual mechanism. STAT3 and STAT6 are aberrantly activated in renal cysts. Genetic and pharmacological approaches to inhibit STAT3 or STAT6 have led to promising results in ADPKD mouse models. Here, we review current findings that lead to a model of PC1 as a key regulator of STAT signaling in renal tubule cells. We discuss how PC1 may orchestrate appropriate epithelial responses to renal injury, and how this system may lead to aberrant STAT activation in ADPKD thereby causing inappropriate activation of tissue repair programs that culminate in renal cyst growth and fibrosis.

Mutations in the gene coding for the integral membrane protein polycystin-1 (PC1) are the cause of most cases of autosomal-dominant polycystic kidney disease (ADPKD), a very common disease that leads to kidney failure and currently lacks approved treatment. Recent work has revealed that PC1 can regulate the transcription factor STAT3, and that STAT3 is aberrantly activated in the kidneys of ADPKD patients and PKD mouse models. Recent approaches to directly inhibit STAT3 in PKD mouse models have been promising. Numerous signaling pathways are known to activate STAT3 and many have long been implicated in the pathogenesis of PKD - such as EGF/EGFR, HGF/c-Met, Src. However, a role of STAT3 in the pathogenesis of PKD had never been considered until now. Here, we review the current findings that suggest that STAT3 is a promising target for the treatment of PKD.

ABSTRACT. Decisions about efficacy and safety of therapeutic proteins (TP) designed to target soluble ligands are made in part by their ex vivo quantification. Ligand binding assays (LBAs) are critical tools in measuring serum TP levels in pharmacokinetic, toxicokinetic, and pharmacodynamic studies. This study evaluated the impact of reagent antibody affinities, assay incubation times, and analytical platform on free or total TP quantitation. An ELISA-based LBA that measures monoclonal anti-sclerostin antibody (TPx) was used as the model system. To determine whether the method measures free or total TPx, the effects of K on , K off , and K D were determined. An 8:1 molar ratio of sclerostin (Scl) to TPx compared to a 1:1 molar ratio produced by rabbit polyclonal antibodies to TPx was required to achieve IC 50 , a measure of TPx interference effectiveness, making it unclear whether the ELISA truly measured free TPx. Kinetic analysis revealed that Scl had a rapid dissociation rate (K off ) from TPx and that capture and detection antibodies had significantly higher binding affinities (K D ) to TPx. These kinetic limitations along with long ELISA incubation times lead to the higher molar ratios (8:1) required for achieving 50% inhibition of TPx. However, a microfluidic platform with the same reagent pairs required shorter incubations to achieve a lower Scl IC 50 molar ratio (1:1). The findings from this study provide the bioanalytical community with a deeper understanding of how reagent and platform selection for LBAs can affect what a particular method measures, either free or total TP concentrations.

Biological activity is a critical quality attribute for biopharmaceuticals, which is accurately measured using an appropriate relative potency bioassay. Developing a bioassay is a complex, rigorous undertaking that needs to address several challenges including modelling all of the mechanisms of action associated with the biotherapeutic. Bioassay development is also an exciting and fast evolving field, not only from a scientific, medical and technological point of view, but also in terms of statistical approaches and regulatory expectations. This has led to an industry-wide discussion on the most appropriate ways to develop, validate and control the bioassays throughout the drug lifecycle.

Autosomal dominant polycystic kidney disease (ADPKD) affects more than 500,000 individuals in the United States alone. In most cases, ADPKD is caused by a loss-of-function mutation in the PKD1 gene, which encodes polycystin-1 (PC1). Previous studies reported that PC1 interacts with atypical protein kinase C (aPKC). Here we show that PC1 binds to the ζ isoform of aPKC (PKCζ) and identify two PKCζ phosphorylation sites on PC1’s C-terminal tail. PKCζ expression is down-regulated in patients with ADPKD and orthologous and nonorthologous PKD mouse models. We find that the US Food and Drug Administration–approved drug FTY720 restores PKCζ expression in in vitro and in vivo models of polycystic kidney disease (PKD) and this correlates with ameliorated disease progression in multiple PKD mouse models. Importantly, we show that FTY720 treatment is less effective in PKCζ null versions of these PKD mouse models, elucidating a PKCζ-specific mechanism of action that includes inhibiting STAT3 activity and cyst-lining cell proliferation. Taken together, our results reveal that PKCζ down-regulation is a hallmark of PKD and that its stabilization by FTY720 may represent a therapeutic approach to the treat the disease.