Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells

Abstract

The tight junctional pore-forming protein claudin-2 (CLDN-2) mediates paracellular Na ؉ and water transport in leaky epithelia and alters cancer cell proliferation. Previously, we reported that tumor necrosis factor-␣ time-dependently alters CLDN-2 expression in tubular epithelial cells. Here, we found a similar expression pattern in a mouse kidney injury model (unilateral ureteral obstruction), consisting of an initial increase followed by a drop in CLDN-2 protein expression. CLDN-2 silencing in LLC-PK 1 tubular cells induced activation and phosphorylation of guanine nucleotide exchange factor H1 (GEF-H1), leading to Ras homolog family member A (RHOA) activation. Silencing of other claudins had no such effects, and re-expression of an siRNA-resistant CLDN-2 prevented RHOA activation, indicating specific effects of CLDN-2 on RHOA. Moreover, kidneys from CLDN-2 knockout mice had elevated levels of active RHOA. Of note, CLDN-2 silencing reduced LLC-PK1 cell proliferation and elevated expression of cyclin-dependent kinase inhibitor P27 (P27KIP1) in a GEF-H1/RHOA-dependent manner. P27KIP1 silencing abrogated the effects of CLDN-2 depletion on proliferation. CLDN-2 loss also activated myocardin-related transcription factor (MRTF), a fibrogenic RHOA effector, and elevated expression of connective tissue growth factor and smooth muscle actin. Finally, CLDN-2 downregulation contributed to RHOA activation and smooth muscle actin expression induced by prolonged tumor necrosis factor-␣ treatment, because they were mitigated by re-expression of CLDN-2. Our results indicate that CLDN-2 suppresses GEF-H1/RHOA. CLDN-2 down-regulation, for example, by inflammation, can reduce proliferation and promote MRTF activation through RHOA. These findings suggest that the initial CLDN-2 elevation might aid epithelial regeneration, and CLDN-2 loss could contribute to fibrotic reprogramming.

Claudin-2: Roles beyond Permeability Functions

“…The effects of various cytokines on claudin-2 were extensively explored. For example, several interleukins, including IL-1β, 6, 9, 13, and 22 control claudin-2, and some were shown to act via the Jak/Stat pathway [65,66,67]. The effect of TNFα might be organ-specific, as it was verified to have different effects on claudin-2 in intestinal and tubular cells (e.g., [53,56]).…”

“…In contrast, ZO family adapters augment the localization of claudin-2 to the TJs. Silencing of ZO-1 and 2 reduced claudin-2 levels, likely in part by enhancing its degradation [52,67,94,95]. Interestingly, however, loss of ZO-1 also inhibited the claudin-2 promoter, suggesting the existence of feedback regulation between claudin-2 turnover and synthesis [52].…”

“…These correlations, however, do not prove a causative connection. More definitive proof is provided by gain- and loss-of-function experiments in a variety of cell lines, showing that primary claudin-2 expression changes can alter proliferation (e.g., [67,103,116]). The experimental evidence connecting claudin-2 with downstream effects is summarized in Table 1.…”

“…Thus, claudin-2 might be a permissive factor for proliferation and may act as a mediator of pro-proliferative signaling. Accordingly, silencing claudin-2 in the colon cancer cell line Caco-2 and in tubular cells prevented EGF- and TNFα-induced increase in cell proliferation [51,67].…”

Measuring Cell Growth and Junction Development in Epithelial Cells Using Electric Cell-Substrate Impedance Sensing (ECIS)

“…This protocol describes the use of ECIS to follow tubular epithelial cell growth, and the development of barrier via formation of the tight junctions. We used this protocol to assess the development of cell confluence (Amoozadeh et al, 2018) and to follow effects of silencing of a tight junction protein, claudin-2 rate on proliferation rate and barrier function (Dan et al, 2019). Silencing claudin-2 alters both barrier function (resulting in elevated resistance) (Amoozadeh et al, 2018) and cell growth (reducing proliferation) (Dan et al, 2019).…”