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Intestinal epithelial self-renewal is a tightly controlled process, which is critically dependent on WNT signalling. Aberrant activation of the WNT pathway in intestinal stem cells (ISCs) results in constitutive transcription of target genes, which collectively drive malignant transformation in colorectal cancer (CRC). However, the contribution of individual genes to intestinal homeostasis and tumorigenesis often is incompletely defined. Here, we discuss converging evidence indicating that the receptor tyrosine kinase (RTK) MET and its ligand hepatocyte growth factor (HGF) play a major role in the intestinal damage response, as well as in intestinal tumorigenesis, by controlling the proliferation, survival, motility, and stemness of normal and neoplastic intestinal epithelial cells. These activities of MET are promoted by specific CD44 isoforms expressed by ISCs. The accrued data indicate that MET and the EGFR have overlapping roles in the biology of intestinal epithelium and that metastatic CRCs can exploit this redundancy to escape from EGFR-targeted treatments, co-opting HGF/MET/CD44v signalling. Hence, targeting both pathways may be required for effective treatment of (a subset of) CRCs. The RTK identity of MET, the distinctive 'plasminogen-like' structure and activation mode of its ligand HGF, and the specific collaboration of MET with CD44, provide several unique targeting options, which merit further exploration., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background & Aims: Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice., Methods: We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (Ah Cre /Met fl/fl /LacZ) or ISC-specific disruption of MET (Lgr5 Creert2 /Met fl/fl /LacZ) and control mice (Ah Cre /Met +/+ /LacZ, Lgr5 Creert2 /Met +/+ /LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5 Creert2 /Met fl/fl /Apc fl/fl and Lgr5 Creert2 /Met +/+ /Apc fl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in Ah Cre /Met fl/fl /Apc fl/+ mice compared with Ah Cre /Met +/+ /Apc fl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44 +/+ , Cd44 -/- , Cd44 s/s , or Cd44 v4-10/v4-10 mice)., Results: Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in Ah Cre /Met fl/fl /LacZ mice. Lgr5 Creert2 /Met fl/fl /LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5 Creert2 /Met fl/fl /Apc fl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (Ah Cre /Met fl/fl /Apc fl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44 -/- mice did not expand to the same extent as crypts from Cd44 +/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10., Conclusions: In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Chronic kidney diseases (CKDs) are characterized by tubular atrophy and interstitial fibrosis. We previously showed that in obstructive nephropathy de novo CD44 renal expression contributes to renal fibrosis but attenuates tubular damage/apoptosis. As CD44-standard (CD44s) has been linked to TGF-β1-mediated actions and CD44-variant-3 (CD44v3) favors HGF-c-Met binding, we compared the functional properties of these CD44 isoforms in the progression of obstructive nephropathy, using specific CD44-variant knockout/knockin mice. The presence of CD44v3 diminished tubular damage during obstructive nephropathy, decreased apoptosis, and increased proliferation of tubular epithelial cells, and prevented renal fibrosis development. In contrast, expression of CD44s led to increased tubular damage and tubular epithelial cell apoptosis, and more renal fibrosis. A relative increase in renal β-catenin expression, HGF production, and HGF/c-Met signaling, together with a relative inhibition of TGF-β1 downstream signaling and TGF-β type I receptor expression, was found in CD44v3 mice compared with CD44s littermates. In line with this, Wnt3a/HGF treatment of tubular cells resulted in higher β-catenin/p-AKT levels in CD44v3(+) tubular epithelial cells, whereas TGF-β1 induced a mild collagen I upregulation in CD44v3(+) mouse embryonic fibroblasts as compared with CD44s(+) cells. Thus, CD44s and CD44v3 exert opposite roles in the progression of obstructive nephropathy, with CD44v3-v10 being the protective isoform that delays evolution of the renal pathology.

Acute kidney injury (AKI) is a common complication during systemic inflammatory response syndrome (SIRS), a potentially deadly clinical condition characterized by whole-body inflammatory state and organ dysfunction. CD44 is a ubiquitously expressed cell-surface transmembrane receptor with multiple functions in inflammatory processes, including sterile renal inflammation. The present study aimed to assess the role of CD44 in endotoxic shock-induced kidney inflammation and dysfunction by using CD44 KO and WT mice exposed intraperitoneally to LPS for 2, 4, and 24 hours . Upon LPS administration, CD44 expression in WT kidneys was augmented at all time-points. At 2 and 4 hours, CD44 KO animals showed a preserved renal function in comparison to WT mice. In absence of CD44, the pro-inflammatory cytokine levels in plasma and kidneys were lower, while renal expression of the anti-inflammatory cytokine IL-10 was higher. The cytokine levels were associated with decreased leukocyte influx and endothelial activation in CD44 KO kidneys. Furthermore, in vitro assays demonstrated a role of CD44 in enhancing macrophage cytokine responses to LPS and leukocyte migration. In conclusion, our study demonstrates that lack of CD44 impairs the early pro-inflammatory cytokine response to LPS, diminishes leukocyte migration/chemotaxis and endothelial activation, hence, delays endotoxic shock-induced AKI.

CD44 marks stem cell-like cells in a number of tumour types, including colorectal cancer (CRC), while aberrant CD44 expression conveys increased tumourigenic, invasive, and metastatic potential. Previous data indicate that CD44 is a direct target of p53-mediated transcriptional repression in breast cancer. Since inactivating p53 mutations are frequent genetic events in CRC these could unleash expression of CD44. In the present study, we therefore explored the relation between p53 mutational status and CD44 expression in a cohort of 90 localized primary CRCs and studied the effect of radiation-induced p53 activation on CD44 expression. Interestingly, we observed that, in contrast to breast cancer, loss of function p53 mutations were not associated with elevated CD44 expression in colon cancer. Moreover, DNA-damage induced p53 activation did not result in repression of CD44 expression, neither in colon cancer cells nor in normal intestinal epithelial cells. Our data demonstrate that CD44 expression in normal and malignant intestinal epithelial cells is not regulated by p53, implying that regulation of this potentially important therapeutic target is tissue and cancer-type specific.

Objective: Lack of the adhesion molecule CD44 reduces collateral artery growth (arteriogenesis) in a murine hindlimb model. CD44 function is influenced by expression of 10 alternatively spliced exons (v1-v10), with unknown effects on arteriogenesis. As the variant exon CD44v3 binds heparan sulphate and facilitates preservation of growth factors, we hypothesized that the variably spliced exon region of CD44, especially exon CD44v3, is involved in arteriogenesis., Materials and Methods: The right femoral artery of C57BL/6J-mice was ligated and tissue was processed for histological and qPCR analysis of CD44-isoform expression. Microsphere perfusion measurements were performed in mice lacking the variably spliced exon region (CD44s knock-in mice), and in knock-in strains with specific isoform expression (CD44v3-10 and CD44v4-10), as well as in double knock-in mice, expressing CD44v3-10 and CD44s., Results: Expression of total CD44 and CD44v3 mRNA following femoral artery ligation was increased, accompanied by increased mRNA levels of the CD44-relevant splicing factors Tra2-beta1 and SRm160. CD44v3-expression was limited to the vessel wall of growing collateral arteries. Perfusion restoration was significantly reduced in mice lacking the variably spliced exon region (CD44s):20.1 ± 1.3%, compared to the background strain: 57.3 ± 2.2%. Mice expressing exon v3 (CD44v3-10) showed perfusion percentages of 25.9 ± 1.1%, compared to mice lacking this exon (CD44v4-10):19.1 ± 0.7%. Combined expression of CD44v3 and CD44s further improved perfusion restoration: 33.1 ± 2.6%., Conclusion: Total CD44 and CD44v3 mRNA are upregulated during arteriogenesis. The absence of the variably spliced exon region impairs arteriogenesis. Presence of exon v3 of CD44 results in improved arteriogenesis. Expression of CD44s and CD44v3 provides a synergistic effect on arteriogenesis. As this combined expression still resulted in hampered arteriogenesis, a specific role of exon v2 in arteriogenesis appears likely.

Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or beta-catenin plays a critical role in the initiation of colorectal cancer. These mutations cause constitutively active beta-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to colorectal cancer precursor lesions, called dysplastic aberrant crypt foci. CD44 is a prominent WNT signaling target in the intestine and is selectively expressed on the renewing epithelial cells lining the crypts. The expression of CD44 is dramatically increased in aberrant crypt foci in both humans and tumor-susceptible Apc(Min/+) mice, suggesting a role for CD44 in intestinal tumorigenesis. To study this role, we crossed C57BL/6J-Cd44(-/-) mice with C57BL/6J-Apc(Min/+) mice. Compared with C57BL/6J-Cd44(+/+)/Apc(Min/+) mice, C57BL/6J-Cd44(-/-)/Apc(Min/+) mice showed an almost 50% reduction in the number of intestinal adenomas. This reduction was primarily caused by a decrease in the formation of aberrant crypts, implying the involvement of CD44 in tumor initiation. The absence of CD44 in the normal (nonneoplastic) crypts of Cd44(-/-)/Apc(Min/+) mice did not alter the proliferative capacity and size of the intestinal stem cell and transit-amplifying compartments. However, compared with Cd44(+/+)/Apc(Min/+) mice, Cd44(-/-)/Apc(Min/+) showed an increase in the number of apoptotic epithelial cells at the base of the crypt which correlated with an increased expression of the proapoptotic genes Bok and Dr6. Our results show an important role for CD44 in intestinal tumorigenesis and suggest that CD44 does not affect proliferation but is involved in the control of the balance between survival and apoptosis in the intestinal crypt.

Escherichia coli is the most common pathogen found in urinary tract infections (UTIs), mainly affecting children and women. We report that CD44, a hyaluronic acid (HA) binding protein that mediates cell-cell and cell-matrix interactions, facilitates the interaction of E. coli with urothelial cells and thus the infection of the host. We found that CD44 is constitutively expressed on urothelial cells and that HA accumulates in E. coli-induced UTI. In CD44-deficient mice, the bacterial outgrowth was dramatically less compared with wild-type mice despite similar granulocyte influx in the bladder and in the kidney as well as comparable cytokines/chemokines levels in both genotypes. E. coli was able to bind HA, which adhered to CD44-positive tubular epithelial cells. Most importantly, the interaction of CD44 on tubular epithelial cells with HA facilitated the migration of E. coli through the epithelial monolayer. The results provide evidence that CD44 on urothelial cells facilitates E. coli UTI. Disruption of the interaction between CD44 and HA in the bladder may provide a new approach to prevent and to treat UTI.

CD44 is a glycoprotein that is involved in inflammation and cell-cell/cell-matrix interactions, is upregulated in the kidney upon injury, and leads to fibrosis through enhancement of TGF-beta1 signaling. Absence of CD44 prevents development of renal fibrosis in unilateral ureteral obstruction (UUO). A hallmark of development of renal fibrosis is the degeneration of the capillary network. This study shows that CD44 is upregulated on capillary endothelial cells during UUO. For elucidation of the role of CD44 on peritubular endothelial cells in UUO, capillary network degeneration was compared in CD44+/+ and CD44-/- mice. As expected, degeneration of the capillary network was observed in CD44+/+ mice during UUO, associated with increased endothelial apoptosis. However, in the absence of CD44, degeneration of the network is prevented as a result of a decrease in the rate of apoptosis in endothelial cells. The divergence in endothelial apoptosis is not correlated to differential vascular endothelial growth factor or thrombospondin-1 expression. For further investigation of capillary regression, CD44+/+ and CD44-/- peritubular capillary endothelial cell lines were established. With the use of these cells, it is shown that interaction between CD44 and its ligand hyaluronic acid enhances the proapoptotic effect of TGF-beta1 but not thrombospondin-1 on endothelial cells, contributing to the degeneration of the capillary network. Blocking interaction between hyaluronic acid and CD44 therefore may be a potential therapeutic opportunity to preserve the capillary network and prevent the development of fibrosis in chronic renal disease.

Inflammation contributes to renal ischemia reperfusion (I/R) injury, potentially causing renal dysfunction. The inflammatory infiltrate mainly consists of neutrophils, which are deleterious for the renal tissue. Because CD44 is expressed by neutrophils and is rapidly upregulated by capillary endothelial cells after I/R injury, it was hypothesized that CD44 might play an important role in the development of I/R injury. This study showed that rapid CD44 upregulation on renal capillary endothelial cells mediates neutrophil recruitment to the postischemic tissue. Hence, CD44 deficiency led to decreased influx of neutrophils regardless of comparable levels in chemotactic factors expressed in the kidney. The reduced influx of neutrophils was associated with preserved renal function and morphology. Adoptive transfer experiments of labeled neutrophils revealed that endothelial CD44 rather than neutrophil CD44 mediates neutrophil migration. Activation of neutrophils increased cell-surface expression of hyaluronic acid (HA). Altogether, a novel mechanism in the recruitment of neutrophils that involves interaction of endothelial CD44 and neutrophil HA was found. Either blocking endothelial CD44 or removal of neutrophil HA decreased rolling and adhesion of neutrophils. Administration of anti-CD44 to mice reduced the influx of neutrophils into the postischemic tissue, associated with renal function preservation. Therefore, anti-CD44-based therapies may contribute to prevent or reduce renal I/R injury.

CD44 is a glycoprotein involved in inflammation and cell-cell/cell-matrix interactions. CD44 is upregulated in the kidney upon injury; however, its role in the pathogenesis of renal damage and fibrosis remains largely unknown. The authors show that mice lacking CD44 developed more tubular damage, associated with decreased proliferation and increased apoptosis of tubular epithelial cells, but less renal fibrosis after unilateral ureteral obstruction. In addition, impaired influx of macrophages and decreased accumulation of myofibroblasts was observed in the obstructed kidney of CD44(-/-) mice compared with CD44(+/+) mice. Hepatocyte growth factor (HGF) and transforming growth factor-beta1 (TGF-beta1) exert reciprocal functions in the progression of renal diseases and interact with CD44 in vitro. For the first time, the authors establish diminished HGF-signaling, via its high affinity receptor c-Met, in the absence of CD44 in vivo. In parallel, the signaling of TGF-beta1 reflected by the relative phosphorylation and nuclear translocation of Smad-2 and Smad-3 was reduced in the obstructed kidney of CD44(-/-) mice. In conclusion, CD44 exerts protective effects on tubuli but contributes to renal fibrogenesis at least in part through enhancement of HGF and TGF-beta1 signaling pathway in obstructive nephropathy.

Cell migration and phagocytosis are both important for controlling Mycobacterium tuberculosis infection and are critically dependent on the reorganization of the cytoskeleton. Since CD44 is an adhesion molecule involved in inflammatory responses and is connected to the actin cytoskeleton, we investigated the role of CD44 in both these processes. Macrophage (Mphi) recruitment into M. tuberculosis-infected lungs and delayed-type hypersensitivity sites was impaired in CD44-deficient (CD44(-/-)) mice. In addition, the number of T lymphocytes and the concentration of the protective key cytokine IFN-gamma were reduced in the lungs of infected CD44(-/-) mice. The production of IFN-gamma by splenocytes of CD44(-/-) mice was profoundly increased upon antigen-specific stimulation. Flow cytometry analysis revealed that soluble CD44 can directly bind to virulent M. tuberculosis. Mycobacteria also interacted with Mphi-associated CD44, as reflected by reduced binding and internalization of bacilli by CD44(-/-) Mphis. This suggests that CD44 is a receptor on Mphis for binding of M. tuberculosis. CD44(-/-) mice displayed a decreased survival and an enhanced mycobacterial outgrowth in lungs and liver during pulmonary tuberculosis. In summary, we have identified CD44 as a new Mphi binding site for M. tuberculosis that mediates mycobacterial phagocytosis, Mphi recruitment, and protective immunity against pulmonary tuberculosis.

We have previously shown, by light microscopy, that the level of expression of CD44 (pan-CD44, CD44v3, CD44v5, and CD44v6) in human basal cell carcinomas is related to growth pattern and invasiveness (Br J Dermatol 1099;140:17-25). We have now studied the fine distribution of these CD44 isoforms in the same tumors using immunoelectron microscopy. Despite the strong differences in the level of expression in tumor areas with different growth patterns, CD44 was consistently found almost exclusively at intercellular surfaces, with a very strong predilection for widened intercellular pouches, ie, identical to the distribution in the normal epidermis. This prevalent distribution corroborates a role for CD44 in maintaining hyaluronan-filled spaces (J Histochem Cytochem 1998;46:241-248). However, the correlation between the presence of CD44 and the presence of such pouches was not absolute, indicating that other factors are involved as well. In contrast to the prevailing literature, we also found a weak but distinct labeling of cell surfaces facing the extracellular matrix. Interestingly, this appeared significantly elevated in the thinnest, most irregular, and usually most peripheral tumor cell strands, where it was associated with tumor cell protrusions and absence of a basal lamina. Thus, the CD44(+) protrusions were in direct contact with the extracellular matrix and apparently represented sites of invasion. The mechanisms that may contribute to a role of CD44 at these sites include binding of extracellular matrix components (notably hyaluronan) and several biologically active factors such as hepatocyte growth factor/scatter factor and matrix metalloproteinase 9.

Immunoglobulin A (IgA) nephropathy is a frequent, chronic renal disease characterized by a broad spectrum of clinical presentations and pathologic findings. CD44, a family of type I transmembrane glycoproteins involved in cell-cell and cell-matrix interactions, may orchestrate partially the cascade of inflammation, accumulation of myofibroblasts, and fibrosis leading to end-stage renal disease. To clarify the possible role of CD44 in the progression of IgA nephropathy, the expression of CD44 in glomeruli and the tubulointerstitial compartment was analyzed in 25 renal biopsy specimens of patients with IgA nephropathy and was correlated to histopathologic, serologic, and urinary parameters. The expression of CD44 correlated significantly with the degree of glomerular and interstitial damage, even better than the accumulation of alpha-smooth muscle actin-positive myofibroblasts, which is recognized as a reliable marker for the progression of IgA nephropathy. A positive correlation also was found between proteinuria and the expression of CD44 in the tubulointerstitial compartment. The glomerular and tubulointerstitial expression of CD44 correlated with the degree of renal damage in IgA nephropathy and could be a reliable marker of the progression of IgA nephropathy. CD44 may have a pivotal role in the cascade of renal inflammation and fibrosis., (Copyright 2002 by the National Kidney Foundation, Inc.)