Amy Reynolds

Dynamic and differential regulation of NKCC1 by calcium and cAMP in the native human colonic epithelium

The capacity of the intestine to secrete fluid is dependent on the basolateral Na+–K+–2Cl− co‐transporter (NKCC1). Given that cAMP and Ca2+ signals promote sustained and transient episodes of fluid secretion, respectively, this study investigated the differential regulation of functional NKCC1 membrane expression in the native human colonic epithelium. Tissue sections and colonic crypts were obtained from sigmoid rectal biopsy tissue samples. Cellular location of NKCC1, Na+–K+‐ATPase, M3 muscarinic acetylcholine receptor (M3AChR) and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide‐sensitive uptake), intracellular Ca2+ and cell volume were assessed by 2′,7′‐bis(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF), Fura‐2 and differential interference contrast/calcein imaging. Unstimulated NKCC1 was expressed on basolateral membranes and exhibited a topological expression gradient, predominant at the crypt base. Cholinergic Ca2+ signals initiated at the crypt base and spread along the crypt axis. In response, NKCC1 underwent a Ca2+‐dependent 4 h cycle of recruitment to basolateral membranes, activation, internalization, degradation and re‐expression. Internalization was prevented by the epidermal growth factor receptor kinase inhibitor tyrphostin‐AG1478, and re‐expression was prohibited by the protein synthesis inhibitor cylcoheximide; the lysosome inhibitor chloroquine promoted accumulation of NKCC1 vesicles. NKCC1 internalization and re‐expression were accompanied by secretory volume decrease and bumetanide‐sensitive regulatory volume increase, respectively. In contrast, forskolin (i.e. cAMP elevation)‐stimulated NKCC1 activity was sustained, and membrane expression and cell volume remained constant. Co‐stimulation with forskolin and acetylcholine promoted dramatic recruitment of NKCC1 to basolateral membranes and prolonged the cycle of co‐transporter activation, internalization and re‐expression. In conclusion, persistent NKCC1 activation by cAMP is constrained by a Ca2+‐dependent cycle of co‐transporter internalization, degradation and re‐expression; this is a novel mechanism to limit intestinal fluid loss.

Canonical Wnt signals combined with suppressed TGFβ/BMP pathways promote renewal of the native human colonic epithelium

Background A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. Objective To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFβ/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. Design Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. Results Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFβ and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFβ or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. Conclusions Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFβ/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population.

Compromised Tissue Renewal in the Ageing Human Colonic Epithelium

BACKGROUND: The intestinal epithelium is the most rapidly renewing tissue in the body. It is widely believed that this attribute minimises the accumulation of age-related molecular damage. Increasing evidence suggests that this protective mechanism is undermined by agerelated molecular changes that accumulate in long-lived stem/progenitor cells. Age-related molecular damage in the intestinal epithelium of flies and rodents manifests as a hyperproliferative state that exhibits a greater degree of apoptosis, clonogen/stem cell number and reduced regenerative potential following damage. Furthermore, the human colonic epithelium is subject to age-related accumulation of mutations in mitochondrial DNA. Given that ageing is a major risk factor for cancer, it is surprising that the status of tissue renewal in the ageing human colonic epithelium has received little attention. AIM: To investigate age-related changes in the renewal of the human colonic epithelium.METHODS: Tissue biopsies obtained at sigmoidoscopy (Ethical approval) from young ( 70 years, N= 12) individuals with no apparent pathology were immediately fixed or processed for crypt isolation. Isolated crypts were observed in 3D culture by digital time-lapse microscopy. Native crypts obtained by microdissection of fixed biopsy tissue were subjected to morphometric analysis and immunofluorescence for detection of Ki67 (proliferation marker), beta catenin, c-Myc and axin2 (all markers for Wnt signals), and OLFM4 (an intestinal stem cell marker). RESULTS: Crypts from young subjects (n=116 crypts) were significantly longer (p< 0.05) than those derived from the older cohort (117 crypts), 375± 10 um versus 330 ± 10 um, respectively. The percentage of Ki67 positive cells in all regions along the cryptaxis was significantly greater (p<0.05) in tissue from older subjects : e.g. crypt base 35% (young) versus 52% (old); mid region 42%% (young) versus 62% (old) and upper region 10% (young) versus 17% (old). The number of cell divisions observed under timelapse microscopy was reduced for crypts derived from older subjects suggesting that the increased Ki67 labelling index in the older group reflected a slower cell cycle time. Although the nature of the proliferating cell type is not known at this stage, an increase in OLFM4+ (stem) cell number was observed along the axis of crypts derived from older subjects. The above traits were associated with a trend towards an extended profile of immunolabelling intensity for nuclear beta catenin and Wnt target gene expression along the crypt-axis of tissue derived from older subjects. CONCLUSIONS: Age-related changes in crypt length, cell proliferation and markers for intestinal stem cells and Wnt signalling components along the crypt-axis suggest that tissue renewal is compromised in the ageing human colonic epithelium.