Global depletion of STAT5 predisposes mice to experimental colitis (Han et al, 2009a
), however, the phenotype could be caused by the defective STAT5 signalling in IEC compartment and/or sub-epithelial laminar propria (Yao et al, 2006
). We have recently shown that STAT5 in enterocytes is required for GM-CSF regulation of homeostatic responses to gut injury (Han et al, 2010
). Thus, in this study, we investigated whether enterocyte STAT5 directly regulates intestinal homeostatic responses to injury. We demonstrated that an IEC-specific stat5
deletion increases the susceptibility of these animals to DSS-induced colitis as well as to NSAID-induced ileal injury in mice (); STAT5IEC KO
mice with colitis exhibited delayed intestinal mucosal wound healing (), as characterized by significantly higher levels of colonic mucosal inflammatory cells, Th1 and Th17 cytokines and NF-κB activation in STAT5IEC KO
mice as compared to STAT5IEC WT
mice after a 5-day recovery period. Consistently, the extended time of DSS treatment and recovery resulted in the worse mucosal injury and poorer healing in STAT5IEC KO
mice (). These extra time points excluded the possibility that it is a particular kinetic of recovery which could affect the mucosal physiology and wound healing in response to gut injury. Accordingly, enterocyte STAT5 is required for intestinal mucosal wound healing in response to gut injury.
TNF-α, IFN-γ and IL-1β induce NF-κB activation in the well-differentiated IECs, leading to a progressive increase in MLCK protein expression. This process has been postulated to be an important mechanism that contributes to intestinal inflammation (Al-Sadi et al, 2009
; Turner, 2009
). Epithelial-specific IκBα mutant transgenic mice exhibit attenuated T cell-induced diarrhea and abrogated increased paracellular permeability (Tang et al, 2010
). These studies suggest that aberrant activation of NF-κB in IECs controls the expression of many inflammatory cytokines involved in the pathogenesis of IBD. In normal mammary gland development and differentiation, NF-κB regulates the milk protein gene β-casein by negatively interfering with STAT5 tyrosine phosphorylation (Geymayer & Doppler, 2000
). Conversely, we reported that STAT5 signalling antagonize NF-κB binding activity to regulate epithelial survival (Han et al, 2006
; Han et al, 2009a
). Our current studies showed that depletion of STAT5 in IECs resulted in reduction of IκBα cytosolic abundance, upregulation of NF-κB nuclear activation and delayed mucosal wound healing. Interestingly, consistent with the recent report (Smyth et al, 2011
), we found that combination of IFN-γ and TNF-α increased STAT5 nuclear abundance in HT-29 monolayers (Supporting information Fig S5C
). We think that simultaneous up-regulation of STAT5 also reflects a feed-back response by IEC monolayer during injury; in other words, the up-regulated STAT5 plays a protective role in the monolayer TJ disintegration induced by cytokine-mediated NFκB and MLCK activation. Together, these data support an interactive regulation between the STAT5 and NF-κB pathways, and this interaction may impact IEC barrier function and regulate mucosal wound repair ().
The disrupted IEC barrier has been proposed to play a critical role in initiating disease through broadly activating mucosal immune responses and accelerating the onset and severity of immune-mediated colitis (Blair et al, 2006
; Su et al, 2009
), for instance, genetic ablation of IL-10 increases small bowel para-cellular permeability, leading to the spontaneous colitis in mice (Madsen et al, 1999
). Conversely, preservation of the integrity of the IEC barrier may promote intestinal mucosal healing in IBD (Dignass, 2001
). In our studies, STAT5IEC KO
mice exhibited IEC barrier dysfunction at baseline, which was characterized by significantly elevated paracellular permeability, focal colonic IEC apoptosis, and slightly increased proliferation; however, knockdown of STAT5 in well-differentiated IEC monolayers did not increase apoptosis under basal conditions. In patients with active IBD, increased IEC apoptosis, erosions and ulcer-type lesions contribute to focal barrier defects. In contrast, morphological breakage and loss of TJ strands are associated with the global barrier defects seen in active IBD (Schulzke et al, 2009
; Zeissig et al, 2007
). Ultrastructural analyses confirmed that the bulk of the transmucosal transport occurred across TJ rather than via gross epithelial defects, such as ulcers (Machen & Erlij, 1972
; Shen et al, 2010
). Therefore, the IEC apoptosis in STAT5IEC KO
mice may be secondary to up-regulated proinflammatory cytokines caused by IEC TJ barrier disruption, infiltrated luminal bacteria, and sequentially activated adaptive immune reaction.
Furthermore, our data demonstrated that NF-κB activation, MLCK mRNA levels and phosphorylation of MLC were upregulated, and that TJ assembly was disrupted in STAT5IEC KO
mice. Among the various components of TJ, ZO-1, -2 and -3 exhibited reduced protein and RNA levels as well as a disrupted distribution. Knockdown of STAT5 activated NF-κB, elevated pMLC levels and reduced ZO abundance, causing monolayer hyperpermeability. ZO-1, -2 and -3 have been shown to localize to the adherens junction (AJ) with cadherin and catenins prior to the localization of other membrane-associated TJPs, such as occludin (Mitic & Anderson, 1998
). ZO proteins regulate both AJ and TJ function by directly binding to F-actin (Yamazaki et al, 2008
). Depletion of both ZO-1 and -2 in the EpH4 mammary epithelial cell line completely abrogates TJ assembly and genetic deletion of ZO genes results in embryonic lethality (Xu et al, 2008
). More importantly, MLCK may regulate TJ barrier function by directly mediating ZO-1 exchange and anchoring (Yu et al, 2010
), and MLC phosphorylation caused ZO-1 redistribution, which can be reversed by MLCK inhibition (Shen & Turner, 2006
). Thus, ZO proteins may play a key role in directing the regulation of TJ assembly. We speculate that STAT5 controls TJ integrity by regulating MLCK-dependent ZO assembly or by direct interaction with ZO proteins; however, our data do not reveal a direct interaction between TJ assembly and STAT5 activity. Taken together, these findings show that STAT5 signalling plays a non-redundant role in ZO expression and assembly.
A constitutively active truncated MLCK (tMLCK)-mediated MLC phosphorylation is sufficient to trigger regulation of TJ barrier function and ZO-1 redistribution in IEC monolayers (Shen et al, 2006
). We found that long form MLCK expression as well as pMLC was elevated in STAT5 deficient and knockdown IEC monolayers under basal conditions, and STAT5 regulated MLCK promoter activity. Conversely, depletion of long MLCK reduced increased IEC para-cellular permeability in STAT5IEC KO
mice. Thus, we speculate that STAT5 controls TJ assembly under basal conditions, partially by regulating MLCK-dependent ZO assembly (STAT5
MLCK → ZOs). We found that, during water recovery, CA-MLCK Tg mice exhibited slower wound healing and repair of intestinal mucosa than littermate controls. Conversely, MLCK KO mice were resistant to DSS-induced mucosal injury. Importantly, STAT5 activation in IECs was abrogated in CA-MLCK Tg mice and restored in long form MLCK KO mice following DSS exposure compared to littermate controls. Therefore, the impaired STAT5 signalling in IECs delayed intestinal epithelial wound healing, and IEC STAT5 signalling is required for repairing mucosal wound induced by loss of IEC barrier. However, our data also suggest that other factors could contribute to the maintenance of increased IEC TJ permeability during mucosal inflammation. The on-going studies with an extension of mouse genotypes will further determine the other factors involved in the intestinal phenotypes due to loss of epithelial STAT5.
To definitively demonstrate that STAT5 regulates NF-κB activity in IECs, we simultaneously knocked down STAT5 and RelA/p65 in HT-29 IEC monolayers and stimulated IEC monolayers with the combination of IFN-γ and TNF-α (Wang et al, 2006
). We observed that knockdown of RelA/p65 prevented the upregulated MLC phosphorylation and TJ hyperpermeability in IEC monolayers, which were induced by either STAT5 knockdown or proinflammatory cytokine administration. Most importantly, we found that loss of STAT5 enhanced the NF-κB and MLCK promoter activity in response to cytokine administration, and that STAT5 antagonizes NF-κB DNA binding in long MLCK promoter. Accordingly, IEC STAT5 may negatively regulate NF-κB activation of MLCK to modulate MLC phosphorylation (STAT5
NFκB → MLCK), which protects IEC barrier function to promote IEC wound healing ().
Taken together, our findings suggest that a novel negative feedback mechanism (STAT5
NFκB → MLCK) directly regulates intestinal epithelial TJ barrier function and immune responses to injury relevant to human IBD (). Our results demonstrate the regulation of epithelial STAT5 activity as a means to improve IEC barrier dysfunction and mucosal inflammation in IBD. Thus, these studies will provide a potential therapeutic target for treating intestinal barrier dysfunction and mucosal wound healing in IBD.