Suppression of TGFß and Angiogenesis by Type VII Collagen in Cutaneous SCC.

BACKGROUND: Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGFß signaling and angiogenesis in vitro and in vivo. METHODS: Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGFß pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided. RESULTS: Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between α2ß1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFß signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFß markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFß markers, n = 6; VEGF, n = 21). Inhibition of TGFß receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siTßRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS: Type VII collagen suppresses TGFß signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.

Generation of point-mutant FAK knockin mice.

Focal adhesion kinase is a non-receptor protein tyrosine kinase with signaling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration, and proliferation it also has non-kinase scaffolding functions in the nucleus. Focal adhesion kinase (FAK) activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signaling of activated FAK elicits changes in cellular behavior. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites, in vitro much less is known about their functions in vivo. Here, we report the generation of a series of FAK-mutant knockin mice where mutant FAK, either kinase dead, non-phosphorylatable mutants Y397F and Y861F, or mutant Y397E-containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre-expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism.

Accelerated re-epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling.

The upregulation of TGF-beta1 and integrin expression during wound healing has implicated these molecules in this process, but their precise regulation and roles remain unclear. Here we report that, notably, mice lacking beta(3)-integrins show enhanced wound healing with re-epithelialization complete several days earlier than in wild-type mice. We show that this effect is the result of an increase in TGF-beta1 and enhanced dermal fibroblast infiltration into wounds of beta(3)-null mice. Specifically, beta(3)-integrin deficiency is associated with elevated TGF-beta receptor I and receptor II expression, reduced Smad3 levels, sustained Smad2 and Smad4 nuclear localization and enhanced TGF-beta1-mediated dermal fibroblast migration. These data indicate that alpha(v)beta(3)-integrin can suppress TGF-beta1-mediated signaling, thereby controlling the rate of wound healing, and highlight a new mechanism for TGF-beta1 regulation by beta(3)-integrins.

Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon.

BACKGROUND AND AIMS: In order to establish whether extraintestinal cells contribute to the turnover and repair of gastrointestinal tissues, we studied the colons and small intestines of female mice that had received a male bone marrow transplant, together with gastrointestinal biopsies from female patients that had developed graft versus host disease after receiving a bone marrow transplant from male donors. METHODS: Using in situ hybridisation to detect Y chromosomes and immunohistochemistry, we demonstrated that cells derived from injected bone marrow frequently engrafted into the intestine and differentiated into pericryptal myofibroblasts. RESULTS: In the human intestine, we confirmed by combining in situ hybridisation with immunostaining for smooth muscle actin that the bone marrow derived cells within the intestine exhibited a myofibroblast phenotype. In female mouse recipients of male bone marrow grafts, we observed colocalisation of Y chromosomes and clusters of newly formed marrow derived myofibroblasts. While few of these were present at seven days after bone marrow transplantation, they were numerous at 14 days, and by six weeks entire columns of pericryptal myofibroblasts could be seen running up the sides of crypts in both the small intestine and colon. These columns appeared to extend into the villi in the small intestine. Within the intestinal lamina propria, these Y chromosome positive cells were negative for the mouse macrophage marker F4/80 antigen and CD34. CONCLUSIONS: Bone marrow derived pericryptal myofibroblasts were present in the mouse intestine following irradiation and bone marrow transplant, and in the intestines of human patients suffering graft versus host disease following a bone marrow transplant. Our data indicate that bone marrow cells contribute to the regeneration of intestinal myofibroblasts and epithelium after damage, and we suggest that this could be exploited therapeutically.

Bone marrow contributes to renal parenchymal turnover and regeneration.

In order to establish whether extra-renal cells contribute to the turnover and repair of renal tissues, this study examined kidneys of female mice that had received a male bone marrow transplant and kidney biopsies from male patients who had received kidney transplants from female donors. By using in situ hybridization to detect Y-chromosomes it could be demonstrated that circulating stem cells frequently engraft into the kidney and differentiate into renal parenchymal cells. In the human renal grafts it was confirmed that some of the recipient-derived cells within the kidney exhibited a tubular epithelial phenotype, by combining in situ hybridization with immunostaining for the epithelial markers CAM 5.2 and the lectin Ulex europaeus. Female mouse recipients of male bone marrow grafts showed co-localization of Y-chromosomes and tubular epithelial markers Ricinus communis and Lens culinaris, and a specific cytochrome P450 enzyme (CYP1A2) indicating an appropriate functional capability of clustered newly formed marrow-derived tubular epithelial cells. Y-chromosome-containing cells were observed within glomeruli, with morphology and location appropriate for podocytes. Within the murine kidney, these Y-chromosome-positive cells were negative for the mouse macrophage marker F4/80 antigen and leukocyte common antigen, but were vimentin-positive. The presence of bone marrow-derived cells was noted in both histologically normal mouse kidneys and in human transplanted kidneys suffering damage from a variety of causes. These data indicate that bone marrow cells contribute to both normal turnover of renal epithelia and regeneration after damage, and it is suggested that this could be exploited therapeutically.

Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts.

Osteoclasts express the alphavbeta3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the beta3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that alphavbeta3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, alphavbeta5, does not substitute for alphavbeta3 during cytokine stimulation or authentic osteoclastogenesis. beta3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, beta3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in beta3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin alphavbeta3 is essential for normal osteoclast function.

Insights and questions arising from studies of a mouse model of Glanzmann thrombasthenia.

In summary, studies of beta 3-deficient mice demonstrate that they provide an excellent model of the human bleeding disorder, Glanzmann thrombasthenia (GT), and replicate essentially all features of the human disease. Furthermore, these mice reveal additional defects consequent upon the absence of integrins alpha v beta 3 and alpha IIB beta 3 that had not become evident from studies of the human patients. These include defects in placental development and in bone resorption. In contrast, despite published evidence suggesting a role for alpha v beta 3 in angiogenesis, beta 3-null mice have not yet shown any defects in this process. The availability of these mice will allow extensive further studies of GT pathogenesis and therapy and of the roles of beta 3 integrins in different forms of angiogenesis, including the pathologically important angiogenic processes in the retina and in response to tumors. Furthermore, they open the way to studies of a variety of other processes in which roles for beta 3 integrins have been suggested.

Integrins in vascular development.

Many growth factors and their protein kinase receptors play a role in regulating vascular development. In addition, cell adhesion molecules, such as integrins and their ligands in the extracellular matrix, play important roles in the adhesion, migration, proliferation, survival and differentiation of the cells that form the vasculature. Some integrins are known to be regulated by angiogenic growth factors and studies with inhibitors of integrin functions and using strains of mice lacking specific integrins clearly implicate some of these molecules in vasculogenesis and angiogenesis. However, the data are incomplete and sometimes discordant and it is unclear how angiogenic growth factors and integrin-mediated adhesive events cooperate in the diverse cell biological processes involved in forming the vasculature. Consideration of the results suggests working hypotheses and raises questions for future research directions.

Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival.

beta3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (alphaIIbbeta3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (alphavbeta3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the alphaIIb or the beta3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of beta3 integrins, we have generated a strain of beta3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of beta3 integrins and we report that postnatal neovascularization of the retina appears to be beta3-integrin-independent, contrary to expectations from inhibition experiments.

Novel roles for alpha3beta1 integrin as a regulator of cytoskeletal assembly and as a trans-dominant inhibitor of integrin receptor function in mouse keratinocytes.

Previously we found that alpha3beta1 integrin-deficient neonatal mice develop micro-blisters at the epidermal-dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of alpha3beta1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of alpha3beta1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of alpha3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and alpha-actinin at focal contact sites in the alpha3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to alpha3beta1 deficiency. Apart from the loss of alpha3beta1 there is no change in expression of the other integrins expressed by the alpha3-null keratinocytes. However, in functional assays, alpha3beta1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of alpha3beta1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.

alpha3beta1 Integrin is required for normal development of the epidermal basement membrane.

Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.