The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin–mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits βcatenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin-mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

Background: Contralateral prophylactic mastectomy (CPM) is being performed with increased frequency. Predictors of CPM and their impact on breast reconstruction are examined.Methods: A retrospective review of a dually trained oncologic and plastic surgeon's experience with patients undergoing total mastectomy from 2002 to 2012 was performed. Patients who underwent bilateral therapeutic mastectomies or who had previous contralateral mastectomy were excluded from this series.Results: Four hundred forty-six patients were treated with total mastectomy and 174 (39%) underwent CPM. The incidence of CPM nearly tripled over the period studied. Compared to women treated with unilateral mastectomy, women who elected for CPM were younger (mean age, 50.4 vs 56.8 years, P < 0.001), leaner (mean body mass index, 26.1 vs 27.4 kg/m(2), P = 0.036), more often white (86.8% vs 73.8%, P = 0.004), and more often had a family history of breast cancer (52% vs 33.3%, P < 0.001). The CPM group was also more likely to have undergone a preoperative magnetic resonance imaging (56.3% vs 39%, P < 0.001) and to have stage I disease (31% vs 22.8%, P = 0.053). They were less likely to have undergone prior attempts at breast conservation (6.9% vs 15.8%, P = 0.004) and considerably more likely to pursue breast reconstruction (83.9% vs 63.6%, P < 0.001). Multivariate analysis confirmed age, white race, family history, prior attempt at breast conservation, and receipt of breast reconstruction to be independently associated with prophylactic mastectomy. Incidental contralateral cancers were discovered in 4% of women who underwent CPM (n = 7), lobular carcinoma in situ in 2.3% (n = 4), and atypical lesions in an additional 11.6% (n = 20). Women who underwent CPM favored reconstruction with breast implants (60.9% vs 17.3%), whereas the transverse rectus abdominis musculocutaneous flap predominated among their unilateral counterparts (38.6% vs 15.5%). Among women who underwent immediate breast reconstruction, the addition of a contralateral procedure expectedly increased breast complication rates (50.3% vs 35.0%, P = 0.007), especially the more severe complications that required hospitalization or reoperation (18.6% vs 5.0%, P < 0.001).Conclusions: The incidence of CPM is increasing and is associated with younger age, white race, family history, and the use of breast reconstruction. Implant-based reconstructions predominate in this cohort. The added morbidity of a contralateral procedure is significant.

The proinflammatory cytokine IFNγ modulates intestinal epithelial (IEC) homeostasis through effects on IEC proliferation. Epithelial proliferative responses are, in turn, regulated by β‐catenin, an adherens junction‐associated protein that differentially controls cell‐cell adhesion and gene transcription. Chronic exposure to IFNγ results in protein kinase B (PKB/Akt)‐mediated β‐catenin signaling that initially promotes, and subsequently suppresses IEC proliferation. The mechanism(s) governing the suppression of β‐catenin signaling are poorly understood. Here we show that the β‐catenin dependent, antiproliferative activity of IFNγ requires synthesis, activation and subcellular compartmentalization of 14.3.3ζ scaffold protein and the Akt isoform Akt1. 14.3.3ζ associates with phosphorylated β‐ catenin (pβ‐cat552) in IECs exposed to IFNγ that in turn promotes β‐catenin dependent‐Akt1 expression. Akt1 upregulation and its accumulation in the nucleus results in translocation of 14.3.3ζ and pβ‐cat552 from the nucleus to the cytosol thereby suppressing β‐ catenin transactivation and IEC proliferation. These results provide a mechanism by which IFNγ decreases active β‐catenin in the nucleus to suppress IEC proliferation during inflammation.

Polymorphonuclear leukocyte (PMN) migration across the intestinal epithelium closely parallels disease symptoms in patients with inflammatory bowel disease. PMN transepithelial migration (TEM) is a multistep process that terminates with PMN detachment from the apical epithelium into the lumen. Using a unique mAb (GM35), we have previously demonstrated that engagement of the CD44 variant containing exon 6 (CD44v6) blocks both PMN detachment and cleavage of CD44v6. In this article, we report that PMN binding to CD44v6 is mediated by protein-specific O-glycosylation with sialyl Lewis A (sLe(a)). Analyses of glycosyltransferase expression identified fucosyltransferase 3 (Fut3) as the key enzyme driving sLe(a) biosynthesis in human intestinal epithelial cells (IECs). Fut3 transfection of sLe(a)-deficient IECs resulted in robust expression of sLe(a). However, this glycan was not expressed on CD44v6 in these transfected IECs; therefore, engagement of sLe(a) had no effect on PMN TEM across these cells. Analyses of sLe(a) in human colonic mucosa revealed minimal expression in noninflamed areas, with striking upregulation under colitic conditions that correlated with increased expression of CD44v6. Importantly, intraluminal administration of mAb GM35 blocked PMN TEM and attenuated associated increases in intestinal permeability in a murine intestinal model of inflammation. These findings identify a unique role for protein-specific O-glycosylation in regulating PMN-epithelial interactions at the luminal surface of the intestine.

Desmosomal cadherins mediate cell–cell adhesion in epithelial tissues and have been known to be altered in cancer. We have previously shown that one of the two intestinal epithelial desmosomal cadherins, desmocollin-2 (Dsc2) loss promotes colonic epithelial carcinoma cell proliferation and tumor formation. In this study we show that loss of the other intestinal desmosomal cadherin, desmoglein-2 (Dsg2) that pairs with Dsc2, results in decreased epithelial cell proliferation and suppressed xenograft tumor growth in mice. Dsg2-deficient cells demonstrated a compensatory increase in Dsc2 expression, and small interfering RNA-mediated loss of Dsc2 restored proliferation in Dsg2-deficient cells. Dsg2 downregulation inhibited epidermal growth factor receptor (EGFR) signaling and cell proliferation through altered phosphorylation of EGFR and downstream extracellular signal-regulated kinase activation in parallel with inhibited EGFR receptor internalization. Additionally, we demonstrated a central role of Dsc2 in controlling EGFR signaling and cell proliferation in intestinal epithelial cells. Consistent with these findings, analyses of human colon cancers demonstrated increased Dsg2 protein expression. Taken together, these data demonstrate that partner desmosomal cadherins Dsg2 and Dsc2 play opposing roles in controlling colonic carcinoma cell proliferation through differential effects on EGFR signaling.

In the next year, approximately 250,000 women will be diagnosed with breast cancer in the United States [1]. The majority of these women will be recommended partial or total mastectomy as a component of their cancer treatment. The goal of breast reconstruction is to rebuild and/or reshape the breast mound to correct the mastectomy defect for physical and psychological reasons. There are a number of reconstructive options available to patients who desire breast reconstruction, including implant-based procedures, autologous tissue reconstructions, or a combination. Additionally, oncoplastic procedures are a recent advance in the field of breast-conserving therapies that further refines our ability to resect breast cancer while preserving the form of the breast. Because planning the reconstruction depends on the type of resection performed and the amount of tissue remaining after mastectomy, the breast surgeon, reconstructive surgeon, and the patient collectively should discuss reconstructive goals and how these can be achieved after the patient's oncologic surgery.

BackgroundRegulated intestinal epithelial cell proliferation and differentiation play a pivotal role in controlling intestinal epithelial homeostasis and barrier function. Pro-inflammatory cytokines such as IFNg and TNFa that are increased in the intestinal mucosa of patients with inflammatory bowel disease (IBD) influence epithelial homeostasis and compromise barrier function. These proinflammatory cytokines modulate intestinal epithelial (IEC) homeostasis through effects on IEC proliferation that is in turn regulated by β-catenin, an adherens junction-associated protein. Chronic exposure to IFNg results in protein kinase B (PKB/Akt)-mediated bcatenin signaling that initially promotes, and subsequently suppresses IEC proliferation. The mechanism(s) governing the suppression of b-catenin signaling after IFNg treatment are however poorly understood and therefore the focus of this study.

Intercellular junction complexes, including adherens junctions and desmosomes (composed of proteins such as desmogleins and E-cadherin), regulate colonic epithelial permeability.Ulcerative colitis (UC) can alter regulation and expression of these proteins.The effect of dysplasia on the expression of these proteins in the background of UC is largely unknown, and the effect on desmoglein-2 has not been well studied.In 7 patients with active UC, 2 biopsies were selected: one showing active UC without dysplasia and another showing active UC with low-grade dysplasia.These were compared with biopsies from 7 different patients showing quiescent UC and 7 normal colon samples.Immunostaining was performed using an antibody against E-cadherin (HECD-1) and 2 antibodies against desmoglein-2 (H-290 [intracellular] and novel AH12.2 [extracellular]).Epithelial immunostaining intensity was scored in each diagnostic group, in dysplasia, and in areas of transmigrating neutrophils.Staining for all 3 antibodies was similar in cases of quiescent UC and normal colon.Staining for all 3 was stronger in active UC than quiescent UC.Transmigrating neutrophils decreased staining intensity for all 3 immunostains in immediately adjacent epithelial cells, compared with active UC without adjacent neutrophils.Dysplasia increased intensity for all 3 stains.Some intensity differences were statistically significant (P = .04for dysplasia vs quiescent UC with AH12.2;P = .0001,.0003,and .02for dysplasia vs normal colon, quiescent UC, and active UC with H-290, respectively; and P = .008and .004for dysplasia vs normal colon and quiescent UC with HECD-1, respectively).Though membrane permeability is increased in active UC, E-cadherin and desmoglein-2 appear up-regulated, perhaps compensating for tight junction functionality loss.Transmigrating neutrophils decrease expression of these proteins, and dysplasia increases expression.These findings illustrate the importance of junction complex molecules and prompt further inquiry regarding their role in UC and other pathologic processes and their potential diagnostic utility.

Expression of the desmosomal cadherins desmoglein‐2 (Dsg2) and desmocollin‐2 (Dsc2) is altered in epithelial cancers, and we have shown that loss of Dsc2 in colonic epithelial cell lines enhances proliferation and tumor formation, thus providing evidence of a tumor suppressor function for Dsc2. To determine if loss of Dsg2 has similar effects, we used shRNA to down‐regulate Dsg2 expression in colonic epithelial cell lines. Interestingly, loss of Dsg2 decreased cell proliferation and inhibited xenograft tumor formation in mice. Furthermore, down‐regulation of Dsg2 in a Dsc2‐negative epithelial cell line had no effect on proliferation or tumor growth, indicating that Dsc2 may promote growth suppression following loss of Dsg2. To examine whether Dsc2 contributes to growth suppression in Dsg2‐deficient cells, we assessed the effect of Dsg2 loss on Dsc2 expression. Our results show that Dsg2‐deficient cells have increased Dsc2 protein levels compared to controls, suggesting that compensatory Dsc2 up‐regulation may mediate growth suppression. Consistent with this notion, siRNA knockdown of Dsc2 restored proliferation in Dsg2‐deficient colonic epithelial cell lines. Taken together, our data demonstrate that, unlike Dsc2, loss of Dsg2 reduces cell growth and inhibits tumor formation in vivo. Thus, anti‐Dsg2‐targeted therapies may be useful for inhibiting growth of tumors expressing both Dsc2 and Dsg2.This work was supported by CCFA Fellowship and AGA Research Scholar Awards (PN) and NIH grants DK61379 (CP), DK55679 and DK59888 (AN).

Desmocollin-2 (Dsc2) and desmoglein-2 (Dsg2) are transmembrane cell adhesion proteins of desmosomes. Reduced expression of Dsc2 has been reported in colorectal carcinomas, suggesting that Dsc2 may play a role in the development and/or progression of colorectal cancer. However, no studies have examined the mechanistic contribution of Dsc2 deficiency to tumorigenesis. Here we report that loss of Dsc2 promotes cell proliferation and enables tumor growth in vivo through the activation of Akt/β-catenin signaling. Inhibition of Akt prevented the increase in β-catenin-dependent transcription and proliferation following Dsc2 knockdown and attenuated the in vivo growth of Dsc2-deficient cells. Taken together, our results provide evidence that loss of Dsc2 contributes to the growth of colorectal cancer cells and highlight a novel mechanism by which the desmosomal cadherins regulate β-catenin signaling.

Transepithelial migration (TEM) of large numbers of neutrophils (PMN) is a histopathological hallmark of many mucosal inflammatory diseases, yet the mechanisms controlling TEM are poorly defined. We have recently reported a role for shedding of CD44v6 during late events in PMN TEM that are associated with detachment from the luminal (apical) membrane. CD44v6 is a highly glycosylated protein, and our studies have revealed that GM35 binding is dependent not only on expression of CD44v6 protein, but also on its appropriate glycosylation. Here we report that PMN-dependent shedding of CD44v6 specifically requires O-linked glycosylation and this shedding event regulates PMN detachment. Furthermore, glycoepitope screening through the Consortium for Functional Glycomics identified sialyl Lewis A (sLeA) as the glycan specifically expressed on CD44v6 that is critical for GM35 function. Such findings were subsequently confirmed in that antibody-mediated masking of sLeA blocked PMN TEM. ELISA analyses revealed that this inhibition of PMN TEM was also accompanied by a blockade of PMN-dependent shedding of epithelial CD44v6. In summary, we have identified a role for sLeA in PMN-associated shedding of the CD44v6 extracellular domain as well as in PMN detachment from the apical intestinal epithelium as a late event in TEM. Increased understanding of these events could provide potential targets for therapeutic interventions. This work is supported by R01 funding (CAP), a CCFA Senior Research Award, and Emory University Research Council seed grant (NAL).

The tight junction (TJ) localizes in the most apical region of the lateral membrane and is essential for regulating the movement of ions and molecules through the paracellular pathway.Additionally, TJ proteins regulate several physiological process including differentiation and proliferation.TJs are formed by a core of transmembrane proteins that are anchored to the actin cytoskeleton via cytosolic plaque proteins.Previously we reported that the transmembrane TJ protein Junctional Adhesion Molecule-A (JAM-A) regulates epithelial proliferation, but the signaling mechanisms underlying this process were unknown.Here, we report that JAM-A controls intestinal epithelial cell proliferation by modulating PI3K/Akt/β-catenin signaling.Using an siRNA approach to down-regulate JAM-A expression in a model intestinal epithelial cell line, we observed that decreased JAM-A expression leads to increased cell proliferation and enhanced transcriptional activity of β-catenin, a known regulator of cell proliferation.In addition, JAM-A deficient cells exhibit increased levels of activated Akt, a serine/threonine kinase that has been previously shown to regulate β-catenin-dependent transcription.Consistent with a role for Akt/β-catenin signaling in our model, we found that Akt-mediated phosphorylation/inhibition of GSK3b (Ser9) was increased in cells lacking JAM-A.Furthermore, Akt inhibition using Triciribine significantly reduces both β-catenin transcriptional activity and cell proliferation in JAM-A deficient cells.In agreement with our In Vitro findings, JAM-A knockout mice display increased cell proliferation and enhanced Akt/β-catenin signaling in the intestinal epithelium.In conclusion these results offer a molecular basis for the link between JAM-A and regulation of proliferation in intestinal epithelial cells.

The desmosomal cadherin desmoglein-2 (Dsg2) is a transmembrane cell adhesion protein that is widely expressed in epithelial and non-epithelial tissues, such as the intestine, epidermis, testis, and heart. Dsg2 has been shown to regulate numerous cellular processes, including proliferation and apoptosis, and we have previously reported that intracellular fragments of Dsg2 promote apoptosis in colonic epithelial cells. While several studies have shown that both the extracellular and intracellular domains of Dsg2 can be targeted by proteases, identification of these putative Dsg2 fragments in colonic epithelial cells has not been performed. Here, we report that the mouse monoclonal antibody (mAb) AH12.2 binds to the first extracellular domain of Dsg2. Using this antibody along with previously described mAb against the extracellular (6D8) and intracellular (DG3.10) domains of Dsg2, we characterize the expression and identify the cleavage fragments of Dsg2 in colonic epithelial cells. This study provides a detailed description of the extracellular and intracellular Dsg2 cleavage fragments that are generated in the simple epithelium of the colon and will guide future studies examining the relationship of these fragments to cellular fate and disease states.

Expression of the tight junction protein junctional adhesion molecule-A (JAM-A) has been linked to proliferation and tumour progression. However, a direct role for JAM-A in regulating proliferative processes has not been shown. By using complementary in vivo and in vitro approaches, we demonstrate that JAM-A restricts intestinal epithelial cell (IEC) proliferation in a dimerization-dependent manner, by inhibiting Akt-dependent β-catenin activation. Furthermore, IECs from transgenic JAM-A(-/-)/β-catenin/T-cell factor reporter mice showed enhanced β-catenin-dependent transcription. Finally, inhibition of Akt reversed colonic crypt hyperproliferation in JAM-A-deficient mice. These data establish a new link between JAM-A and IEC homeostasis.

This chapter contains sections titled: Tight Junctions Desmosomes Future Perspectives References

Intestinal mucosal inflammation in inflammatory bowel disease (IBD) is associated with perturbed intestinal epithelial homeostasis (proliferation/differentiation) and barrier function that are mediated, in part, by pro‐inflammatory cytokines such as IFNγ. However, the mechanisms governing IFNγ‐mediated regulation of epithelial homeostasis (proliferation/differentiation) and barrier function are not understood. Since a master regulator of epithelial homeostasis is E‐cadherin we investigated if IFNγ modulates epithelial homeostasis by regulating signaling proteins downstream of intestinal epithelial cadherins that also regulate cell proliferation. Using complementary in vitro and in vivo approaches, we identified protein kinase B (PKB/Akt) as a key protein that regulates stability of b‐catenin down‐stream of Ecadherin. IFNγ but not TNFα was observed to initially promote phosphorylation and activation of Akt and β‐catenin resulting in a burst of cell proliferation. However, sustained exposure to IFNγ induced stabilization of E‐cadherin and β‐catenin that was dependent on the cytoplasmic scaffold protein, 14‐3‐3ζ and Akt resulted in inhibition of β‐catenin induced cell proliferation. These results provide a novel paradigm through which sustained exposure to IFNγ results in inhibition β‐catenin signaling, reduced cell proliferation and barrier function in IBD. This work was supported by CCFA Fellowship and AGA Research Scholar Awards (PN) and NIH grants DK61379 (CP), DK55679 and DK59888 (AN).

The migration of polymorphonuclear leukocytes (PMNs) across the intestinal epithelium is a histopathological hallmark of many mucosal inflammatory diseases including inflammatory bowel disease. The terminal transmigration step is the detachment of PMNs from the apical surface of the epithelium and their subsequent release into the intestinal lumen. The current study sought to identify epithelial proteins involved in the regulation of PMN migration across intestinal epithelium at the stage at which PMNs reach the apical epithelial surface. A panel of Abs reactive with IFN-γ–stimulated T84 intestinal epithelial cells was generated. Screening efforts identified one mAb, GM35, that prevented PMN detachment from the apical epithelial surface. Microsequencing studies identified the GM35 Ag as human CD44. Transfection studies confirmed this result by demonstrating the loss of the functional activity of the GM35 mAb following attenuation of epithelial CD44 protein expression. Immunoblotting and immunofluorescence revealed the GM35 Ag to be an apically expressed v6 variant exon-containing form of human CD44 (CD44v6). ELISA analysis demonstrated the release of soluble CD44v6 by T84 cells during PMN transepithelial migration. In addition, the observed release of CD44v6 was blocked by GM35 treatment, supporting a connection between CD44v6 release and PMN detachment. Increased expression of CD44v6 and the GM35 Ag was detected in inflamed ulcerative colitis tissue. This study demonstrates that epithelial-expressed CD44v6 plays a role in PMN clearance during inflammatory episodes through regulation of the terminal detachment of PMNs from the apical epithelial surface into the lumen of the intestine.