Keratinocyte Growth Factor Improves Epithelial Structure and Function in a Mouse Model of Intestinal Ischemia/Reperfusion

Background

Intestinal ischemia/reperfusion (I/R) induces the desquamation of the intestinal epithelium, increases the intestinal permeability, and in patients often causes fatal conditions including sepsis and multiple organ failure. Keratinocyte growth factor (KGF) increases intestinal growth, although little is known about KGF activity on intestinal function after intestinal I/R. We hypothesized that KGF administration would improve the intestinal function in a mouse model of intestinal I/R.

Methods

Adult C57BL/6J mice were randomized to three groups: Sham, I/R group and I/R+KGF group. Mice were killed on day 5, and the small bowel was harvested for histology, wet weight, RNA and protein content analysis. Epithelial cell (EC) proliferation was detected by immunohistochemistry for PCNA, and apoptosis was determined by TUNEL staining. The expressions of Claudin-1 and ZO-1 were detected by immunohistochemistry. Epithelial barrier function was assessed with transepithelial resistance (TER).

Results

KGF significantly increased the intestinal wet weight, contents of intestinal protein and RNA, villus height, crypt depth and crypt cell proliferation, while KGF resulted in the decrease of epithelial apoptosis. KGF also stimulated the recovery of mucosal structures and attenuated the disrupted distribution of TJ proteins. Moreover, KGF attenuated the intestinal I/R-induced decrease in TER and maintained the intestinal barrier function.

Conclusion

KGF administration improves the epithelial structure and barrier function in a mouse model of intestinal I/R. This suggests that KGF may have clinical applicability.     

IL-1 activates the Na+/H+ antiport in a murine T cell

One of the early events following growth factor exposure is elevation of intracellular pH, a process mediated by the Na+/H+ antiport. We studied the effects of human rIL-1 alpha (HrIL-1 alpha) on intracellular pH (pHi) and calcium ([Ca2+]i) in a murine T cell line (MD10 cells), which proliferates in response to IL-1 alone. By using the intracellularly trapped fluorescent dyes (2(1),7(1)-bis-2-carboxyethyl)-5(and -6) carboxyfluorescein) and indo-1, we monitored immediate to early changes of pHi and [Ca2+]i in response to HrIL-1 alpha. Exposure to HrIL-1 alpha (120 pM) leads to an early, sustained intracellular alkalinization (delta pH = + 0.09 +/- 0.03) that plateaus within 20 min. Lower concentrations of the monokine (12 pM, 1.2 pM) have a positive but not statistically significant effect on pHi. These effects parallel the degree of MD10 IL-1R saturation predicted by the KD (49 pM) as assessed by 125I-HrIL-1 alpha binding by MD10 cells (Bmax = approximately 1300). Both the MD10 IL-1 receptor KD and the HrIL-1 alpha concentration required to induce early measurable alkaline pH shifts, however, exceed by three orders of magnitude the HrIL-1 alpha ED50 (50 fM) required for MD10 proliferation. The IL-1-induced rise in pHi is both sodium dependent and amiloride sensitive, indicative of activation of the Na+/H+ antiport. Additionally, PMA (100 nM) and IL-2 (2 nM) alkalinize MD10 cells, with the rise in pHi as a result of PMA exceeding the maximal IL-1 effect (delta pH = + 0.13 +/- 0.04). Furthermore, although PMA alkalinizes cells previously exposed to HrIL-1 alpha, the monokine does not alter the pHi of PMA-treated MD10 cells. Importantly, intracellular alkalinization induced by either HrIL-1 alpha or PMA is inhibited by staurosporine (1 mu iM). Finally, HrIL-1 alpha does not change MD10 [Ca2+]i, in either an acute or sustained fashion. These results indicate that IL-1 activates the Na+/H+ antiport in T cells by a mechanism that is unrelated to changes in [Ca2+]i but may involve protein kinase C activation.     

Bcl10 links saturated fat overnutrition with hepatocellular NF-kB activation and insulin resistance

Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. With high-fat feeding, FFAs activate NF-kB in target tissues, initiating negative crosstalk with insulin signaling. However, the mechanisms underlying FFA-dependent NF-kB activation remain unclear. Here, we demonstrate that the saturated FA, palmitate, requires Bcl10 for NF-kB activation in hepatocytes. Uptake of palmitate, metabolism to diacylglycerol, and subsequent activation of protein kinase C (PKC) appear to mechanistically link palmitate with Bcl10, known as a central component of a signaling complex that, along with CARMA3 and MALT1, activates NF-kB downstream of selected cell surface receptors. Consequently, Bcl10-deficient mice are protected from hepatic NF-kB activation and insulin resistance following brief high-fat diet, suggesting that Bcl10 plays a major role in the metabolic consequences of acute overnutrition. Surprisingly, while CARMA3 also participates in the palmitate response, MALT1 is completely dispensable, thereby revealing an apparent nonclassical role for Bcl10 in NF-kB signaling.     

Vav3 regulates osteoclast function and bone mass

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.     

Oophorectomy-induced bone loss is attenuated in MAGP1-deficient mice

Microfibril-associated glycoprotein-1 (MAGP1), together with the fibrillins, are constitutive components of vertebrate microfibrils. Mice deficient in MAGP1 (murine MAGP1 knockout animals (Mfap2(-/-)); MAGP1Δ) is appropriate develop progressive osteopenia and reduced whole bone strength, and have elevated numbers of osteoclasts lining the bone surface. Our previous studies suggested that the increased osteoclast population was associated with elevated levels of receptor activator of NF-κB ligand (RANKL), a positive regulator of osteoclast differentiation. To explore the relationship between RANKL expression and osteoclast differentiation in MAGP1 deficiency, oophorectomy (OVX) was used to stimulate RANKL expression in both WT and MAGP1Δ animals. Bone loss following OVX was monitored using whole body DEXA and in vivo μCT. While WT mice exhibited significant bone loss following OVX, percent bone loss was reduced in MAGP1Δ mice. Further, serum RANKL levels rose significantly in OVX WT mice, whereas, there was only a modest increase in RANKL following OVX in the mutant mice due to already high baseline levels. Elevated RANKL expression was normalized when cultured MAGP1Δ osteoblasts were treated with a neutralizing antibody targeting free TGFβ. These studies provide support for increased RANKL expression associated with MAGP1 deficiency and provide a link to altered TGF-β signaling as a possible causative signaling pathway regulating RANKL expression in MAGP1Δ osteoblasts.     

The influence of one-carbon metabolism on gene promoter methylation in a population-based breast cancer study

Abnormal methylation in gene promoters is a hallmark of the cancer genome; however, factors that may influence promoter methylation have not been well elucidated. As the one-carbon metabolism pathway provides the universal methyl donor for methylation reactions, perturbation of this pathway might influence DNA methylation and, ultimately, affect gene functions. Utilizing approximately 800 breast cancer tumor tissues from a large population-based study, we investigated the relationships between dietary and genetic factors involved in the one-carbon metabolism pathway and promoter methylation of a panel of 13 breast cancer-related genes. We found that CCND2, HIN1 and CHD1 were the most "dietary sensitive" genes, as methylation of their promoters was associated with intakes of at least two out of the eight dietary methyl factors examined. On the other hand, some micronutrients (i.e., B 2 and B 6) were more "epigenetically active" as their intake levels correlated with promoter methylation status in 3 out of the 13 breast cancer genes evaluated. Both positive (hypermethylation) and inverse (hypomethylation) associations with high micronutrient intake were observed. Unlike what we saw for dietary factors, we did not observe any clear patterns between one-carbon genetic polymorphisms and the promoter methylation status of the genes examined. Our results provide preliminary evidence that one-carbon metabolism may have the capacity to influence the breast cancer epigenome. Given that epigenetic alterations are thought to occur early in cancer development and are potentially reversible, dietary modifications may offer promising venues for cancer intervention and prevention.     

Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin

The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.