JAM-A signals through the Hippo pathway to regulate intestinal epithelial proliferation.

JAM-A is a tight-junction-associated protein that contributes to regulation of intestinal homeostasis. We report that JAM-A interacts with NF2 and LATS1, functioning as an initiator of the Hippo signaling pathway, well-known for regulation of proliferation. Consistent with these findings, we observed increased YAP activity in JAM-A-deficient intestinal epithelial cells (IEC). Furthermore, overexpression of a dimerization-deficient mutant, JAM-A-DL1, failed to initiate Hippo signaling, phenocopying JAM-A-deficient IEC, whereas overexpression of JAM-A-WT activated Hippo signaling and suppressed proliferation. Lastly, we identify EVI1, a transcription factor reported to promote cellular proliferation, as a contributor to the pro-proliferative phenotype in JAM-A-DL1 overexpressing IEC downstream of YAP. Collectively, our findings establish a new role for JAM-A as a cell-cell contact sensor, raising implications for understanding the contribution(s) of JAM-A to IEC proliferation in the mammalian epithelium.

Longitudinal Functional Study of Murine Aging: A Resource for Future Study Designs.

Aging is characterized by systemic declines in tissue and organ functions. Interventions that slow these declines represent promising therapeutics to protect against age-related disease and improve the quality of life. In this study, several interventions associated with lifespan extension in invertebrates or improvement of age-related disease were tested in mouse models to determine if they were effective in slowing tissue aging in a broad spectrum of functional assays. Benzoxazole, which extends the lifespan of Caenorhabditis elegans, slowed age-related femoral bone loss in mice. Rates of change were established for clinically significant parameters in untreated mice, including kyphosis, blood glucose, body composition, activity, metabolic measures, and detailed parameters of skeletal aging in bone. These findings have implications for the study of preclinical physiological aging and therapies targeting aging. Finally, an online application was created that includes the calculated rates of change and that enables power and variance to be calculated for many clinically important metrics of aging with an emphasis on bone. This resource will help in future study designs employing novel interventions in aging mice. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo.

CD47 is a ubiquitously expressed transmembrane glycoprotein that regulates inflammatory responses and tissue repair. Here, we show that normal mice treated with anti-CD47 antibodies, and Cd47-null mice have impaired intestinal mucosal wound healing. Furthermore, intestinal epithelial cell (IEC)-specific loss of CD47 does not induce spontaneous immune-mediated intestinal barrier disruption but results in defective mucosal repair after biopsy-induced colonic wounding or Dextran Sulfate Sodium (DSS)-induced mucosal damage. In vitro analyses using primary cultures of CD47-deficient murine colonic IEC or human colonoid-derived IEC treated with CD47-blocking antibodies demonstrate impaired epithelial cell migration in wound healing assays. Defective wound repair after CD47 loss is linked to decreased epithelial ß1 integrin and focal adhesion signaling, as well as reduced thrombospondin-1 and TGF-ß1. These results demonstrate a critical role for IEC-expressed CD47 in regulating mucosal repair and raise important considerations for possible alterations in wound healing secondary to therapeutic targeting of CD47.

Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair.

Mucosal wound repair is coordinated by dynamic crosstalk between endogenous and exogenous mediators and specific receptors on epithelial cells and infiltrating immune cells. One class of such receptor-ligand pairs involves formyl peptide receptors (FPRs) that have been shown to influence inflammatory response and repair. Here we explored the role of murine Fpr2/3, an ortholog of human FPR2/receptor for lipoxin A4 (ALX), in orchestrating intestinal mucosal repair. Compared with wild-type (WT) mice, Fpr2/3-/- mice exhibited delayed recovery from acute experimental colitis and perturbed repair after biopsy-induced colonic mucosal injury. Decreased numbers of infiltrating monocytes were observed in healing wounds from Fpr2/3-/- mice compared with WT animals. Bone marrow transplant experiments revealed that Fpr2/3-/- monocytes showed a competitive disadvantage when infiltrating colonic wounds. Moreover, Fpr2/3-/- monocytes were defective in chemotactic responses to the chemokine CC chemokine ligand (CCL)20, which is up-regulated during early phases of inflammation. Analysis of Fpr2/3-/- monocytes revealed altered expression of the CCL20 receptor CC chemokine receptor (CCR)6, suggesting that Fpr2/3 regulates CCL20-CCR6-mediated monocyte chemotaxis to sites of mucosal injury in the gut. These findings demonstrate an important contribution of Fpr2/3 in facilitating monocyte recruitment to sites of mucosal injury to influence wound repair.-Birkl, D., O'Leary, M. N., Quiros, M., Azcutia, V., Schaller, M., Reed, M., Nishio, H., Keeney, J., Neish, A. S., Lukacs, N. W., Parkos, C. A., Nusrat, A. Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair.

IFN-γ-tethered hydrogels enhance mesenchymal stem cell-based immunomodulation and promote tissue repair.

Because of their immunomodulatory activities, human mesenchymal stem cells (hMSCs) are being explored to treat a variety of chronic conditions such as inflammatory bowel disorders and graft-vs-host disease. Treating hMSCs with IFN-γ prior to administration augments these immunomodulatory properties; however, this ex vivo treatment limits the broad applicability of this therapy due to technical and regulatory issues. In this study, we engineered an injectable synthetic hydrogel with tethered recombinant IFN-γ that activates encapsulated hMSCs to increase their immunomodulatory functions and avoids the need for ex vivo manipulation. Tethering IFN-γ to the hydrogel increases retention of IFN-γ within the biomaterial while preserving its biological activity. hMSCs encapsulated within hydrogels with tethered IFN-γ exhibited significant differences in cytokine secretion and showed a potent ability to halt activated T-cell proliferation and monocyte-derived dendritic cell differentiation compared to hMSCs that were pre-treated with IFN-γ and untreated hMSCs. Importantly, hMSCs encapsulated within hydrogels with tethered IFN-γ accelerated healing of colonic mucosal wounds in both immunocompromised and immunocompetent mice. This novel approach for licensing hMSCs with IFN-γ may enhance the clinical translation and efficacy of hMSC-based therapies.

TNFα promotes mucosal wound repair through enhanced platelet activating factor receptor signaling in the epithelium.

Pathobiology of several chronic inflammatory disorders, including ulcerative colitis and Crohn's disease is related to intermittent, spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). In this report, we show that TNFα promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor, Src and Rac1 signaling to promote wound closure. Consistent with these findings, delayed colonic mucosal repair was observed after administration of a neutralizing TNFα antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa, the pro-inflammatory milieu containing TNFα and PAF sets the stage for reparative events mediated by PAFR signaling.

Serum Amyloid A1 Is an Epithelial Prorestitutive Factor.

Several proteins endogenously produced during the process of intestinal wound healing have demonstrated prorestitutive properties. The presence of serum amyloid A1 (SAA1), an acute-phase reactant, within inflamed tissues, where it exerts chemotaxis of phagocytes, is well recognized; however, a putative role in intestinal wound repair has not been described. Herein, we show that SAA1 induces intestinal epithelial cell migration, spreading, and attachment through a formyl peptide receptor 2-dependent mechanism. Induction of the prorestitutive phenotype is concentration and time dependent and is associated with epithelial reactive oxygen species production and alterations in p130 Crk-associated substrate staining. In addition, using a murine model of wound recovery, we provide evidence that SAA1 is dynamically and temporally regulated, and that the elaboration of SAA1 within the wound microenvironment correlates with the influx of SAA1/CD11b coexpressing immune cells and increases in cytokines known to induce SAA expression. Overall, the present work demonstrates an important role for SAA in epithelial wound recovery and provides evidence for a physiological role in the wound environment.

mTORC1 Activation during Repeated Regeneration Impairs Somatic Stem Cell Maintenance.

The balance between self-renewal and differentiation ensures long-term maintenance of stem cell (SC) pools in regenerating epithelial tissues. This balance is challenged during periods of high regenerative pressure and is often compromised in aged animals. Here, we show that target of rapamycin (TOR) signaling is a key regulator of SC loss during repeated regenerative episodes. In response to regenerative stimuli, SCs in the intestinal epithelium of the fly and in the tracheal epithelium of mice exhibit transient activation of TOR signaling. Although this activation is required for SCs to rapidly proliferate in response to damage, repeated rounds of damage lead to SC loss. Consistently, age-related SC loss in the mouse trachea and in muscle can be prevented by pharmacologic or genetic inhibition, respectively, of mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight an evolutionarily conserved role of TOR signaling in SC function and identify repeated rounds of mTORC1 activation as a driver of age-related SC decline.

Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling.

In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

α-Lipoic acid treatment prevents cystine urolithiasis in a mouse model of cystinuria.

Cystinuria is an incompletely dominant disorder characterized by defective urinary cystine reabsorption that results in the formation of cystine-based urinary stones. Current treatment options are limited in their effectiveness at preventing stone recurrence and are often poorly tolerated. We report that the nutritional supplement α-lipoic acid inhibits cystine stone formation in the Slc3a1-/- mouse model of cystinuria by increasing the solubility of urinary cystine. These findings identify a novel therapeutic strategy for the clinical treatment of cystinuria.

Ribosomal Proteins Rpl22 and Rpl22l1 Control Morphogenesis by Regulating Pre-mRNA Splicing.

Most ribosomal proteins (RP) are regarded as essential, static components that contribute only to ribosome biogenesis and protein synthesis. However, emerging evidence suggests that RNA-binding RP are dynamic and can influence cellular processes by performing "extraribosomal," regulatory functions involving binding to select critical target mRNAs. We report here that the RP, Rpl22, and its highly homologous paralog Rpl22-Like1 (Rpl22l1 or Like1) play critical, extraribosomal roles in embryogenesis. Indeed, they antagonistically control morphogenesis through developmentally regulated localization to the nucleus, where they modulate splicing of the pre-mRNA encoding smad2, an essential transcriptional effector of Nodal/TGF-ß signaling. During gastrulation, Rpl22 binds to intronic sequences of smad2 pre-mRNA and induces exon 9 skipping in cooperation with hnRNP-A1. This action is opposed by its paralog, Like1, which promotes exon 9 inclusion in the mature transcript. The nuclear roles of these RP in controlling morphogenesis represent a fundamentally different and paradigm-shifting mode of action for RP.

Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse.

Cellular senescence suppresses cancer by irreversibly arresting cell proliferation. Senescent cells acquire a proinflammatory senescence-associated secretory phenotype. Many genotoxic chemotherapies target proliferating cells nonspecifically, often with adverse reactions. In accord with prior work, we show that several chemotherapeutic drugs induce senescence of primary murine and human cells. Using a transgenic mouse that permits tracking and eliminating senescent cells, we show that therapy-induced senescent (TIS) cells persist and contribute to local and systemic inflammation. Eliminating TIS cells reduced several short- and long-term effects of the drugs, including bone marrow suppression, cardiac dysfunction, cancer recurrence, and physical activity and strength. Consistent with our findings in mice, the risk of chemotherapy-induced fatigue was significantly greater in humans with increased expression of a senescence marker in T cells prior to chemotherapy. These findings suggest that senescent cells can cause certain chemotherapy side effects, providing a new target to reduce the toxicity of anticancer treatments. SIGNIFICANCE: Many genotoxic chemotherapies have debilitating side effects and also induce cellular senescence in normal tissues. The senescent cells remain chronically present where they can promote local and systemic inflammation that causes or exacerbates many side effects of the chemotherapy. Cancer Discov; 7(2); 165-76. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.

Rapamycin Reverses Metabolic Deficits in Lamin A/C-Deficient Mice.

The role of the mTOR inhibitor, rapamycin, in regulation of adiposity remains controversial. Here, we evaluate mTOR signaling in lipid metabolism in adipose tissues of Lmna-/- mice, a mouse model for dilated cardiomyopathy and muscular dystrophy. Lifespan extension by rapamycin is associated with increased body weight and fat content, two phenotypes we link to suppression of elevated energy expenditure. In both white and brown adipose tissue of Lmna-/- mice, we find that rapamycin inhibits mTORC1 but not mTORC2, leading to suppression of elevated lipolysis and restoration of thermogenic protein UCP1 levels, respectively. The short lifespan and metabolic phenotypes of Lmna-/- mice can be partially rescued by maintaining mice at thermoneutrality. Together, our findings indicate that altered mTOR signaling in Lmna-/- mice leads to a lipodystrophic phenotype that can be rescued with rapamycin, highlighting the effect of loss of adipose tissue in Lmna-/- mice and the consequences of altered mTOR signaling.

Muscle-specific 4E-BP1 signaling activation improves metabolic parameters during aging and obesity.

Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) is a key downstream effector of mTOR complex 1 (mTORC1) that represses cap-dependent mRNA translation initiation by sequestering the translation initiation factor eIF4E. Reduced mTORC1 signaling is associated with life span extension and improved metabolic homeostasis, yet the downstream targets that mediate these benefits are unclear. Here, we demonstrated that enhanced 4E-BP1 activity in mouse skeletal muscle protects against age- and diet-induced insulin resistance and metabolic rate decline. Transgenic animals displayed increased energy expenditure; altered adipose tissue distribution, including reduced white adipose accumulation and preserved brown adipose mass; and were protected from hepatic steatosis. Skeletal muscle-specific 4E-BP1 mediated metabolic protection directly through increased translation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and enhanced respiratory function. Non-cell autonomous protection was through preservation of brown adipose tissue metabolism, which was increased in 4E-BP1 transgenic animals during normal aging and in a response to diet-induced type 2 diabetes. Adipose phenotypes may derive from enhanced skeletal muscle expression and secretion of the known myokine FGF21. Unlike skeletal muscle, enhanced adipose-specific 4E-BP1 activity was not protective but instead was deleterious in response to the same challenges. These findings indicate that regulation of 4E-BP1 in skeletal muscle may serve as an important conduit through which mTORC1 controls metabolism.

The ribosomal protein Rpl22 controls ribosome composition by directly repressing expression of its own paralog, Rpl22l1.

Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22(-/-) mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22(-/-) mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.

Late-life rapamycin treatment reverses age-related heart dysfunction.

Rapamycin has been shown to extend lifespan in numerous model organisms including mice, with the most dramatic longevity effects reported in females. However, little is known about the functional ramifications of this longevity-enhancing paradigm in mammalian tissues. We treated 24-month-old female C57BL/6J mice with rapamycin for 3 months and determined health outcomes via a variety of noninvasive measures of cardiovascular, skeletal, and metabolic health for individual mice. We determined that while rapamycin has mild transient metabolic effects, there are significant benefits to late-life cardiovascular function with a reversal or attenuation of age-related changes in the heart. RNA-seq analysis of cardiac tissue after treatment indicated inflammatory, metabolic, and antihypertrophic expression changes in cardiac tissue as potential mechanisms mediating the functional improvement. Rapamycin treatment also resulted in beneficial behavioral, skeletal, and motor changes in these mice compared with those fed a control diet. From these findings, we propose that late-life rapamycin therapy not only extends the lifespan of mammals, but also confers functional benefits to a number of tissues and mechanistically implicates an improvement in contractile function and antihypertrophic signaling in the aged heart with a reduction in age-related inflammation.

Establishing Rps6 hemizygous mice as a model for studying how ribosomal protein haploinsufficiency impairs erythropoiesis.

Diamond-Blackfan anemia is a congenital hypoproliferative macrocytic anemia and 5q- syndrome myelodysplastic syndrome is an acquired hypoproliferative macrocytic anemia. Their common erythroid phenotype reflects a shared pathophysiology-haploinsufficiency of one of many ribosomal proteins and somatic deletion of one allele of the ribosomal protein S14 gene, respectively. Although these abnormalities lead to defective ribosome biogenesis, why ribosomal protein hemizygosity results in anemia is not certain. Here, we characterize the hematopoietic phenotype of mice lacking one allele of the ribosomal protein S6 gene. The mice have an erythroid phenotype similar to both Diamond-Blackfan anemia and the 5q- syndrome and lenalidomide therapy improves their anemia.