Abstract
Transforming growth factor (TGF)-β signaling in humans is stringently regulated to prevent excessive TGF-β signaling. In tumors, TGF-β signaling can both negatively and positively regulate tumorigenesis dependent on tumor type, but the reason for these opposite effects is unclear. TGF-β signaling is mainly mediated via the Smad-dependent pathway, and herein we found that PDZK1-interacting protein 1 (PDZK1IP1) interacts with Smad4. PDZK1IP1 inhibited both the TGF-β and the bone morphogenetic protein (BMP) pathways without affecting receptor-regulated Smad (R-Smad) phosphorylation. Rather than targeting R-Smad phosphorylation, PDZK1IP1 could interfere with TGF-β- and BMP-induced R-Smad/Smad4 complex formation. Of note, PDZK1IP1 retained Smad4 in the cytoplasm of TGF-β-stimulated cells. To pinpoint PDZK1IP1's functional domain, we created several PDZK1IP1 variants and found that its middle region, from Phe40 to Ala49, plays a key role in its Smad4-regulating activity. PDZK1IP1 knockdown enhanced the expression of the TGF-β target genes Smad7 and prostate transmembrane protein androgen-induced (TMEPAI) upon TGF-β stimulation. In contrast, PDZK1IP1 overexpression suppressed TGF-β-induced reporter activities, cell migration, and cell growth inhibition. In a xenograft tumor model in which TGF-β was previously shown to elicit tumor-promoting effects, PDZK1IP1 gain of function decreased tumor size and increased survival rates. Taken together, these findings indicate that PDZK1IP1 interacts with Smad4 and thereby suppresses the TGF-β signaling pathway.
Highlights
Transforming growth factor (TGF)- signaling in humans is stringently regulated to prevent excessive TGF- signaling
We investigated whether PDZK1IP1 affects the TGF- signaling using the TGF-/activin–induced Smad-driven transcriptional (CAGA)12-luc reporter [27]
Because Smad4 is required for the TGF-/Smad pathway and the bone morphogenetic protein (BMP)/Smad pathway, the (SBE)4-luc reporter, which is activated by BMP, was transfected together with PDZK1IP1 in HepG2 cells to investigate whether PDZK1IP1 perturbs the BMP signaling
Summary
We investigated whether PDZK1IP1 affects the TGF- signaling using the TGF-/activin–induced Smad-driven transcriptional (CAGA)12-luc reporter [27]. To confirm their interaction further, we transiently introduced PDZK1IP1/HA into A549 cells and stained the cells with anti-Smad (green) and anti-HA (red) antibodies Their colocalization could be detected in the cytosol with or without TGF- stimulation. When we introduced PDZKI1P1 to NCI-H290 cells (Fig. 7A), TGF-–induced Smad expression decreased, whereas phosphorylation of Smad upon TGF- stimulation was not altered as seen in Fig. 2A (Fig. 7, B and C). Kaplan–Meier analysis revealed that more than 50% of the mice implanted with NCI-H290 cells carrying PDZK1IP1 could survive for longer than 30 days, whereas all of the mice implanted with mock NCI-H290 cells died within 30 days of implantation (Fig. 7F) These results indicated that PDZK1IP1 can inhibit TGF- signaling to suppress the growth of NCI-H290 mesothelioma cells because NCI-H290 cells might proliferate via endogenous paracrine and/or autocrine secretion of TGF-
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