Abstract TGF-βs are cytokines that signal through the receptor complex of type I and type II receptors. Hemidesmosome (BP180, BP230, plectin/HD1, α6β4 integrin), anchoring filaments (laminin 5), and anchoring fibrils (collagen VII) form a hemidesmosomal adhesion complex that provides stable adherence of keratinocytes to the epidermal basement membrane. Nidogen, collagen IV, and laminins are components of the basement membrane, integrins are cell adhesion molecules, and tenascin-C is a matrix protein. The expression of TGF-β receptors I and II was studied in normal epidermis and lesional and non-lesional psoriatic epidermis by immunohistochemistry. TGF-β1 and TGF-β2 in suction blister fluid and serum were determined by enzyme-linked immunoassay. Suction blister fluid and serum samples were obtained from acne patients before and after oral isotretinoin treatment. Suction blister fluid samples were also obtained from healthy volunteers in two age groups from a control site and a betamethasone-pretreated site. The expression of BP180, BP230, plectin/HD1, α6 integrin, β4 integrin, laminin 5, collagen VII, collagen IV, nidogen, laminin α3 chain, and laminin β1g1 chains was studied in uninvolved dermatitis herpetiformis skin by the immunofluorescence technique. The ultrastructure of the hemidesmosomal inner plaque was studied in uninvolved dermatitis herpetiformis skin by electron microscopy. The suction blister method was used to study intact blisters, open wounds (=blister roofs removed right after blister induction) and calcipotriol-pretreated open wounds in healthy volunteers. The reepithelialization rate and the expression of BP180, BP230, plectin/HD1, β4 integrin, laminin 5, collagen VII, laminin α5 chain, laminin β1 chain, tenascin-C, αvβ5 integrin, β5 integrin, α5 integrin, and α9 integrin during reepithelialization were studied by haematoxylin and eosin stainings and the immunofluorescence technique. BP180, BP230, and plectin/HD1 expression were analyzed by body site to exclude regional variation. In normal epidermis, TGF-β receptors I and II were detected in the basal epidermis. Diffusion calculations suggest that circulation is likely to be a major source of TGF-β for TGF-β receptors in the basal epidermis. Downregulation of TGF-β receptors I and II was seen in lesional psoriatic epidermis, suggesting that hyperproliferating lesional epidermis may have lost TGF-β-mediated growth inhibition. Isotretinoin did not affect the serum TGF-β1 or TGF-β2 levels, but caused a 19% local increase in suction blister fluid TGF-β1. Betamethasone caused a 17% decrease in suction blister fluid TGF-β1, presumably due to glucocorticoid-induced vasoconstriction. Modulation of the interstitial fluid TGF-β1 concentration may be one mechanism by which isotretinoin and betamethasone mediate their effects in skin. Immunoreactivity for BP230 and plectin/HD1 was decreased in the basement membrane zone in uninvolved dermatitis herpetiformis skin in a significant proportion of the patients, suggesting distinct molecular changes in BP230 and plectin/HD1. This may be a factor contributing to blister formation. Reepithelialization rate was considerably slower in intact blisters than in open wounds and was not affected by calcipotriol. BP230 and plectin/HD1 appeared earlier in intact blisters than in open wounds. Reepithelialization took place on a continuous laminin sheath in intact blisters, but the laminin sheath in open wounds was partially discontinuous. It was a novel finding that integrin αvβ5 and integrin β5 antibodies showed divergent distributions in regenerating epidermis. The present results suggest that, in some bullous diseases, removal of the blister roof could accelerate blister healing, calcipotriol treatment does not delay wound epithelialization, a continuous laminin sheath may inhibit reepithelialization, and the formation of the hemidesmosomal inner plaque at the leading edge takes place earlier in the more slowly reepithelializing intact blisters than in open wounds.

Cancer metastasis is a multi-stage process initiated by the cancer cell acquiring the ability to migrate. The protein profile of such a cell undergoes dramatic changes including changes in integrin expression. Integrins play a major role in cell adhesion, motility, differentiation, blood clotting, tissue organization and cell growth as well as cancer cell migration, invasion and metastasis. Integrin a6, which can pair with integrin b4 or b1 is a laminin receptor and is detected in epithelial cells. Earlier studies have reported uPA mediated integrin a6 cleavage in prostate cancer resulting in loss of the ligand binding domain. Site-directed mutagenesis studies have identified the cleavage site to be at R594R595 located in the "stalk" region of the integrin a6. Prostate cancer cells PC3N-a6-RR cells, bearing a R594R595 to A594A595 mutation, engineered to express the uncleavable form of integrin a6 were found to migrate 6.4 folds lesser on Laminin-1 as compared to the PC3N-a6-WT cells which expressed the wild-type integrin a6. This result suggests that integrin a6 cleavage enhances migration. Prostate cancer is known to metastasize to the bone. Injection of the PC3N-a6-WT cells in mouse femurs resulted in increased bone destruction and pain behavior when compared to the femurs injected with PC3N-a6-RR cells indicating that the integrin a6 cleavage could affect and modify the bone microenvironment. An observation that complete conversion of integrin a6 to a6p was not observed in cell lines even in presence of excess uPA suggested a regulatory mechanism. Integrins are known to associate with many proteins including tetraspanins, which are transmembrane proteins, that function as protein adapters. Integrin a6 was found to be refractory to uPA mediated cleavage when complexed with tetraspanin CD151. The amount of integrin a6 available for cleavage increased when CD151 levels were decreased by CD151 siRNA treatment. These results suggest that the integrin a6 available and unavailable for cleavage can be modulated by interaction with CD151 and hence affect the migratory potential of the cell. Collectively these data suggest that integrin a6 cleavage can enhance cell migration, initiate signals to modify the tumor microenvironment and can be regulated by interaction with tetraspanin CD151.

It is well established that failure of the lamellar basement membrane (BM) occurs during the development of equine laminitis. This is due to loss of the crucial BM components; laminins and collagens along with loss of attachment complex, the hemidesmosome, of the basal cell to the underlying BM. Previous studies have suggested that Ln-332 may be the primary protein involved in lamellar failure. However, the details of the progression and mechanism involved in this pathology are not currently fully known. This thesis aimed to refine the proteolytic processes and mechanisms occurring during the development of oligofructose induced laminitis. Through the use of novel temporal lamellar biopsies obtained during the development of laminitis induction, it was determined that loss of both Ln-332 and collagen type IV occurs as early as 12 hours post induction. This loss of reactivity initially occurred in a focal pattern with increasing loss as the disease progressed in severity. At the later stages of laminitis, separation of the basal epithelial cell from the dermal tissue was also observed, however at these points the BM still appeared intact. This suggests that more than one mechanism may be involved in disease pathology; one resulting in fragmentation of the BM while a second results in loss of the cell attachment allowing the intact BM to slip away. Immunohistochemical analysis of lamellar tissue revealed a unique pattern of reactivity for the Ln-332 γ2 antibody D4B5, in which no reactivity was observed in normal lamellar tissue, yet the epitope recognized by this antibody becomes apparent during disease development. This initially led to the hypothesis that cleavage of the γ2 subunit and the release of biologically active fragments may occur. However, at the molecular level, no γ2 fragments were detected by western blotting. In vitro cleavage of partially purified equine Ln-332 revealed that both MMP-2 and MT1-MMP were able to process the molecule to produce fragments corresponding to the biologically active counterparts. This suggests that the change in reactivity with this antibody may be due to other mechanisms such as decreased interaction of Ln-332 with other BM components resulting in loss of structural stability of the BM allowing for a change in the orientation of Ln-332. Increased MMP-2, MMP-9 and MT1-MMP expression has been demonstrated in laminitis and this was assumed to be the causative agent resulting in tissue destruction and failure. However, work in this thesis found no increase in gene expression of MMP- 2 and MT1-MMP, as well as no activation of pro MT1-MMP. Increased pro MMP-9 gene and protein expression was observed early in the disease progression yet no MMP- 9 activation occurred. Additionally, activation of MMP-2 was found to occur late in laminitis progression at least 12 hours following BM degradation, thus MMP-2 activation is a secondary effect of laminitis development. Thus, other proteases are expected to result in BM processing. Gene expression of the metalloprotease ADAMTS-4, was observed to increase early during laminitis development, suggesting this is a putative factor involved in intensifying the degradation of the lamellar BM. Work in this thesis also revealed that both Ln-332 and collagen type IV are widely distributed throughout organs in the equine body and localized primarily to BM structures. A novel finding of this thesis is that not only does BM degradation occur in the lamellar BM, it also occurs in organs remote from the hoof. At both the onset of lameness and the acute phase of laminitis, fragmentation of both Ln-332 and collagen type IV also occurs in both the skin and stomach. Recent studies have indicated that both leukocyte emigration and increased cytokine expression occurs in the lamellar tissue during laminitis. Work in this thesis added to this knowledge as leukocyte infiltration into the lamellar tissue occurs early during oligofructose laminitis induction as does increased IL-6 gene expression. Overall, work conducted in this thesis has added to the knowledge of the events occurring during laminitis development. Even though the complete mechanism of tissue destruction and lamellar failure was not established, the progression of events is now more clear in that BM degradation is one of the first events to occur, while MMP-2 activation occurs secondarily. Thus, other mechanisms must be at work early during laminitis development and discovering what they are must remain a research priority for the realization of effective therapeutic strategies.