Stable Knockdown of Polycystin-1 Confers Integrin-α2β1–Mediated Anoikis Resistance

Abstract

The mechanisms of action of polycystin-1 (PC1) have been difficult to dissect because of its interaction with multiple factors, the heterogeneity of the genetic mutations, and the complexity of the experimental animal models. Here, stable knockdown of PC1 in MDCK epithelial cells was achieved by lentiviral-mediated delivery of a specific small interfering RNA for PKD1. The reduction of PC1 expression prevented tubulogenesis in three-dimensional collagen type I culture in response to hepatocyte growth factor and induced formation of cysts. PC1 knockdown created a condition of haploinsufficiency that led to hyperproliferation, increased adhesion to collagen type I, and increased apoptosis. It was shown that the suppression of PC1 was associated with the increased expression of integrin-␣2␤1 and reduced apoptosis in cells grown on collagen type I. The engagement of integrin-␣2␤1 seemed to be essential for the survival because PC1 knockdown cells were significantly less susceptible to anoikis by a mechanism that was reversible by anti-integrin-␣2␤1 blocking antibodies. Overall, these data link integrin-␣2␤1 to some of the biologic functions that are ascribed to PC1 and establish the potential of this approach for the direct study of PC1 functions in a genetically defined background. Furthermore, these findings indicate that reduction of PC1 expression levels, rather than the loss of heterozygosity, may be sufficient to induce cystogenesis.J Am Soc Nephrol 17: 3049 -3058, 20063049 -3058, . doi: 10.1681 A lterations in the PKD1 and PKD2 genes account for cases of autosomal dominant polycystic kidney disease (ADPKD) with a relative frequency of approximately 85 and 15%, respectively (1-3). PKD1 encodes polycystin-1 (PC1), a large 4302-amino acid membrane protein that participates in multiprotein complexes that are involved in cell-matrix and cell-cell interactions and signaling (4,5). Through the heterodimerization with PC2, a nonselective cationic channel that is encoded by the PKD2 gene, PC1 can function as a gating receptor to control calcium influx (6). The interdependent functions of PC1 and PC2 in kidney are supported by the overlapping ADPKD phenotypic manifestation that is caused by mutations of either gene, although the severity and the progression of the disease are exacerbated when PKD1 is involved. The co-localization of the polycystins in primary cilia suggests their involvement in a cellular mechanosensory system (7). These findings are supported by the evidence that polycystic renal phenotypes arise from the mutation or deletion of various ciliary and adhesion proteins (8 -10). To date, however, it has been difficult to define clearly the functions of PC1 and the molecular mechanisms of cystogenesis that it controls. In part, this difficulty derives from the heterogeneity of the genetic mutations that are identified in patients with ADPKD as well as from the lack of functionally characterized mutants. Furthermore, most of the animal models for polycystic kidney share phenotypic manifestations, but some ...

Polycystin-1 Protein Level Determines Activity of the Gα12/JNK Apoptosis Pathway

“…MDCK cells with silenced PC1 (confirmed by reverse transcription-PCR) (supplemental Fig. 1) show a small increase in apoptosis when grown on plastic (8). Fig.…”

“…PC1-silenced MDCK cells and controls were provided by G. Luca Gusella (Mount Sinai School of Medicine, New York). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 50 units/ml penicillin, 50 g/ml streptomycin (Invitrogen), 2 mM glutamine, 250 ng/ml amphotericin as described previously (8). G␣ 12 -silenced MDCK cells were established by stable expression of short hairpin RNA, compared with green fluorescent protein short hairpin RNA control cells established in parallel (described in Ref.…”

“…Cell culture studies with overexpressed and silenced PC1 also support the concept that decreased or increased levels of PC1 lead to abnormal signaling. For example, overexpression of PC1 in Madin-Darby canine kidney (MDCK) cells leads to decreased apoptosis (7), and silencing of PC1 leads to increased apoptosis due to reduced cell adhesion (anoikis) (8).…”

Primary cilia dynamics instruct tissue patterning and repair of corneal endothelium

“…1). To specifically inactivate IFT88 in the CE, we injected the anterior chamber of 5-to 6-d old mice with 2 μL of VIRHD/E/siLuc or VIRHD/E/siIFT88 lentivectors expressing an siRNA hairpin against luciferase (control) or the murine IFT88 gene, respectively (27). Transduction rates in vivo and cell-type specificity were monitored by injecting the VVPW/H2B-cherry lentivirus expressing cherry-tagged histone H2B under the constitutive CMV promoter.…”

Aberrant Expression of Polycystin-1 in Renal Cell Tumors

“…At the cellular level, a number of molecular anomalies were described in both sporadic RCCs and tumors related to various cystic kidney lesions including over-expression, of c-myc, bcl-2, cmet, and c-Ets oncoproteins as well as deregulated expression of growth factors (12)(13)(14)(15)(16). In this regard delineation of specific molecular processes that lead to transforming events in acquired polycystic kidney but not in ADPKD or autosomal recessive polycystic kidney disease (ARPKD) may reveal other functions of polycystin-1, such as the tumor suppressor activity (17).…”