BACKGROUND

Microvesicles (MVs) are anuclear fragments of cells released from the endosomal compartment or shed from surface membranes. We and other investigators demonstrated that MVs released by mesenchymal stem cells (MSCs) were as effective as the cells themselves in inflammatory injuries, such as after endotoxin-induced acute lung injury. However, the therapeutic effects of MVs in an infectious model of acute lung injury remain unknown.

OBJECTIVE

We investigated the effects of human MSC MVs on lung inflammation, protein permeability, bacterial clearance, and survival after severe bacterial pneumonia.

METHODS

We tested the effects of MVs derived from human MSCs on Escherichia coli pneumonia in mice. We also studied the interactions between MVs and human monocytes and human alveolar epithelial type <em>2</em> cells.

RESULTS

Administration of MVs derived from human MSCs improved survival in part through keratinocyte growth factor secretion and decreased the influx of inflammatory cells, cytokines, protein, and bacteria in mice injured with bacterial pneumonia. In primary cultures of human monocytes or alveolar type <em>2</em> cells, the uptake of MVs was mediated by CD44 receptors, which were essential for the therapeutic effects. MVs enhanced monocyte phagocytosis of bacteria while decreasing inflammatory cytokine secretion and increased intracellular ATP levels in injured alveolar epithelial type <em>2</em> cells. Prestimulation of MSCs with a toll-like receptor 3 agonist further enhanced the therapeutic effects of the released MVs.

CONCLUSIONS

MVs derived from human MSCs were as effective as the parent stem cells in severe bacterial pneumonia.

Glycosaminoglycan-modified isoforms of CD44 have been implicated in <em>growth</em> <em>factor</em> presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding <em>growth</em> <em>factors</em>, basic-fibroblast <em>growth</em> <em>factor</em> (b-FGF), heparin binding-epidermal <em>growth</em> <em>factor</em> (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from <em>keratinocytes</em>, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding <em>growth</em> <em>factors</em> to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. <em>Keratinocytes</em>, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-<em>2</em>)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by <em>keratinocytes</em> in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding <em>growth</em> <em>factors</em> to leukocytes on the vascular cell wall.

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca(<em>2</em>+)-dependent cell adhesion molecules that is first expressed in the epidermis as <em>keratinocytes</em> transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports <em>keratinocyte</em> differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting <em>keratinocyte</em> differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal <em>growth</em> <em>factor</em> receptor-Erk1/<em>2</em> (extracellular signal-regulated kinase 1/<em>2</em>) signaling, thereby facilitating <em>keratinocyte</em> progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

Age-related thymopoietic insufficiency has been proposed to be related to either defects in lymphohematopoietic progenitors or the thymic microenvironment. In this study, we examined whether <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), an epithelial cell-specific <em>growth</em> <em>factor</em>, could increase thymopoietic capacity in aged mice by restoration of the function of thymic epithelial cells (TECs). The thymic cellularity in KGF-treated aged mice increased about 4-fold compared to placebo-treated mice, resulting in an equivalent thymic cellularity to young mice. Enhanced thymopoiesis was maintained for about <em>2</em> months after a single course of KGF, and sustained improvement was achieved by administration of monthly courses of KGF. With the enhanced thymopoiesis after KGF treatment, the number of naive CD4 T cells in the periphery and T-cell-dependent antibody production improved in aged mice. KGF induced increased numbers of TECs and intrathymic interleukin-7 (IL-7) production and reorganization of cortical and medullary architecture. Furthermore, KGF enhanced thymopoiesis and normalized TEC organization in klotho (kl/kl) mice, a model of premature degeneration and aging, which displays thymopoietic defects. The result suggests that TEC damage is pathophysiologically important in thymic aging, and KGF therapy may be clinically useful in improving thymopoiesis and immune function in the elderly.

Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix macromolecules, including interstitial and basement membrane collagens, fibronectin, laminin, and proteoglycan core protein. The enzymes are secreted or released in latent form and become activated in the pericellular environment by disruption of a Zn(++)-cysteine bond which blocks the reactivity of the active site. The major cell types in inflamed and healthy periodontal tissues (fibroblasts, <em>keratinocytes</em>, endothelial cells, and macrophages) are capable of responding to <em>growth</em> <em>factors</em> and cytokines, as well as to products released from the microbial flora by induction of transcription of 1 or more MMP genes. Cytokines that are likely to regulate expression of MMP genes in periodontal tissues include IL-1, TNF-alpha, and TGF-alpha. In addition, triggered PMN leukocytes which express only <em>2</em> MMP (PMN-CL and Mr 9<em>2</em>K GL) release these enzymes from specific granule storage sites in response to a number of stimuli. The evidence that MMP are involved in tissue destruction in human periodontal diseases is still indirect and circumstantial. Cells isolated from normal and inflamed gingiva are capable of expressing a wide complement of MMP in culture and several MMP can be detected in cells of human gingiva in vivo. In addition, PMN-CL and Mr 9<em>2</em>K GL are readily detected in gingival crevicular fluid from gingivitis and periodontitis patients. Osteoclastic bone resorption does not appear to directly involve MMP, but a body of evidence suggests that bone resorption is initiated by removal of the osteoid layer by osteoblasts by means of a collagenase-dependent process.

Smooth muscle cells, macrophages, glial cells, <em>keratinocytes</em>, and transformed cells have been established as synthesis sites for vascular endothelial <em>growth</em> <em>factor</em> (VEGF). The modulating effects of VEGF are essentially limited to endothelial cells (ECs), the only cell type consistently shown to express VEGF receptors. VEGF has thus been considered to act exclusively via a paracrine pathway. We sought to determine whether the role of human ECs might, under selected conditions, extend beyond that of a target to involve contingency synthesis of VEGF. In both unstimulated human umbilical vein ECs (HUVECs) and human derma-derived microvascular ECs (HMECs), Northern analysis detected no VEGF transcripts. Phorbol-1<em>2</em>-myristate 13-acetate (10(-7) M) treatment, however, induced VEGF mRNA expression in both HUVECs and HMECs, peaking at 3 and 6 h, respectively, and returning to undetectable levels by 1<em>2</em> h. In vitro exposure of HUVECs to a hypoxic environment (pO<em>2</em> = 35 mm of mercury) for 1<em>2</em>, <em>2</em>4, and 48 h and exposure of HMECs for 6, 1<em>2</em>, <em>2</em>4, and 48 h induced VEGF mRNA in a time-dependent fashion. Re-exposure to normoxia (pO<em>2</em> = 150 mm of mercury) for <em>2</em>4 h after <em>2</em>4 h of hypoxia returned VEGF mRNA transcripts to undetectable levels in HUVECs. Cobalt chloride and nickel chloride treatment each induced VEGF mRNA in ECs. Cycloheximide treatment further augmented expression of VEGF mRNA induced by cobalt chloride, nickel chloride, and hypoxia in HUVECs. VEGF protein production in hypoxia HUVECs was demonstrated immunohistochemically. Conditioned media from hypoxic HUVECs caused a <em>2</em>-fold increase in the incorporation of tritiated thymidine. Finally, immune precipitates of anti-KDR probed with anti-Tyr(P) antibodies demonstrated evidence of receptor autophosphorylation in hypoxic but not normoxic HUVECs. These findings thus establish the potential for an autocrine pathway that may augment and/or amplify the paracrine effects of VEGF in stimulating angiogenesis.

BACKGROUND

Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta.

RESULTS

MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF, FGF-7), a member of the fibroblast <em>growth</em> <em>factor</em> (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the <em>2</em> potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis.

CONCLUSIONS

Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury.

The Smad<em>2</em> and Smad3 (Smad<em>2</em>/3) proteins are principally involved in the transmission of transforming <em>growth</em> <em>factor</em> beta (TGF-beta) signaling from the plasma membrane to the nucleus. Many transcription <em>factors</em> have been shown to cooperate with the Smad<em>2</em>/3 proteins in regulating the transcription of target genes, enabling appropriate gene expression by cells. Here we identified 1,787 Smad<em>2</em>/3 binding sites in the promoter regions of over <em>2</em>5,500 genes by chromatin immunoprecipitation on microarray in HaCaT <em>keratinocytes</em>. Binding elements for the v-ets erythroblastosis virus E<em>2</em>6 oncogene homolog (ETS) and transcription <em>factor</em> AP-<em>2</em> (TFAP<em>2</em>) were significantly enriched in Smad<em>2</em>/3 binding sites, and knockdown of either ETS1 or TFAP<em>2</em>A resulted in overall alteration of TGF-beta-induced transcription, suggesting general roles for ETS1 and TFAP<em>2</em>A in the transcription induced by TGF-beta-Smad pathways. We identified novel Smad binding sites in the CDKN1A gene where Smad<em>2</em>/3 binding was regulated by ETS1 and TFAP<em>2</em>A. Moreover, we showed that small interfering RNAs for ETS1 and TFAP<em>2</em>A affected TGF-beta-induced cytostasis. We also analyzed Smad<em>2</em>- or Smad3-specific target genes regulated by TGF-beta and found that their specificity did not appear to be solely determined by the amounts of the Smad<em>2</em>/3 proteins bound to the promoters. These findings reveal novel regulatory mechanisms of Smad<em>2</em>/3-induced transcription and provide an essential resource for understanding their roles.

The closure of skin wounds is essential for resistance against microbial pathogens, and <em>keratinocyte</em> migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces <em>keratinocyte</em> migration. In this study, we found that 1 microg/ml LL-37 induced the maximum level of <em>keratinocyte</em> migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) after 10 min, which suggests that LL-37-induced <em>keratinocyte</em> migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. <em>2</em><em>2</em>5 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced <em>keratinocyte</em> migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. <em>2</em><em>2</em>5, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced <em>keratinocyte</em> migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology <em>2</em>-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced <em>keratinocyte</em> migration. Transfection with SOCS1/Jak<em>2</em> binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced <em>keratinocyte</em> migration. In conclusion, LL-37 induces <em>keratinocyte</em> migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak <em>2</em> binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of <em>keratinocyte</em> migration.

Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H(<em>2</em>)O(<em>2</em>) potently induces vascular endothelial <em>growth</em> <em>factor</em> (VEGF) expression in human <em>keratinocytes</em>. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp -194 to -50 of the VEGF promoter is responsible for the H(<em>2</em>)O(<em>2</em>) response. It was established that at microm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible <em>factor</em> (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human <em>keratinocytes</em>. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.

Under normal conditions, homeostasis determines whether a cell remains quiescent, proliferates, differentiates, or undergoes apoptosis. In this state of homeostasis, <em>keratinocytes</em> control melanocyte <em>growth</em> and behaviour through a complex system of paracrine <em>growth</em> <em>factors</em> and cell-cell adhesion molecules. Alteration of this delicate homeostatic balance and can lead to altered expression of cell-cell adhesion and cell communication molecules and to the development of melanoma. Melanoma cells escape from this control by <em>keratinocytes</em> through three major mechanisms: (1) down-regulation of receptors important for communication with <em>keratinocytes</em> such as E-cadherin, P-cadherin, desmoglein and connexins, which is achieved through <em>growth</em> <em>factors</em> produced by fibroblasts or <em>keratinocytes</em>; (<em>2</em>) up-regulation of receptors and signalling molecules not found on melanocytes but important for melanoma-melanoma and melanoma-fibroblast interactions such as N-cadherin, Mel-CAM, and zonula occludens protein-1 (ZO-1); (3) loss of anchorage to the basement membrane because of an altered expression of the extracellular-matrix binding integrin family. In the current review, we describe the alterations in cell-cell adhesion and communication associated with melanoma development and progression, and discuss how a greater understanding of these processes may aid the future therapy of this disease.

The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia, AQP4 in airway epithelia, and AQP5 at the apical plasma membrane in type I cells of alveolar epithelia. We previously studied the role of AQP1 and AQP4 in lung fluid transport using knockout mice. Here, we examined the role of AQP5 using AQP5 knockout mice, which were recently shown to manifest defective saliva secretion. AQP5 deletion did not affect lung morphology at the light microscopic level, nor did it affect the distribution or expression of aquaporins 1, 3, or 4. Airspace-capillary osmotic water permeability (P(f)) was measured in isolated perfused lungs by pleural surface fluorescence and gravimetric methods. P(f) was reduced 10-fold by AQP5 deletion and was further reduced by <em>2</em>- to 3-fold in AQP1/AQP5 double-knockout mice. Hydrostatic lung edema in response to acute increases in pulmonary artery pressure was not affected by AQP5 deletion. Active alveolar fluid absorption was measured in an in situ lung model from the increase in concentration of a volume marker in an isosmolar alveolar instillate. Interestingly, fluid absorption did not differ in litter-matched AQP5 knockout mice, nor was there an effect of AQP5 deletion when fluid absorption was maximally stimulated by pretreatment of mice with <em>keratinocyte</em> <em>growth</em> <em>factor</em>. These results indicate that AQP5 is responsible for the majority of water transport across the apical membrane of type I alveolar epithelial cells. The unimpaired alveolar fluid clearance in AQP5-null mice indicates that high alveolar water permeability is not required for active, near-isosmolar fluid transport.

BACKGROUND

Bone-marrow derived mesenchymal stem cells (MSCs) reduce the severity of evolving acute lung injury (ALI), but their ability to repair the injured lung is not clear. A study was undertaken to determine the potential for MSCs to enhance repair after ventilator-induced lung injury (VILI) and elucidate the mechanisms underlying these effects.

METHODS

Anaesthetised rats underwent injurious ventilation which produced severe ALI. Following recovery, they were given an intravenous injection of MSCs (<em>2</em>×10(6) cells) or vehicle immediately and a second dose <em>2</em>4 h later. The extent of recovery following VILI was assessed after 48 h. Subsequent experiments examined the potential for non-stem cells and for the MSC secretome to enhance VILI repair. The contribution of specific MSC-secreted mediators was then examined in a wound healing model.

RESULTS

MSC therapy enhanced repair following VILI. MSCs enhanced restoration of systemic oxygenation and lung compliance, reduced total lung water, decreased lung inflammation and histological lung injury and restored lung structure. They attenuated alveolar tumour necrosis factor α concentrations while increasing concentrations of interleukin 10. These effects were not seen with non-stem cells (ie, rat fibroblasts). MSC-secreted products also enhanced lung repair and attenuated the inflammatory response following VILI. The beneficial effect of the MSC secretome on repair of pulmonary epithelial wounds was attenuated by prior depletion of keratinocyte growth factor.

CONCLUSIONS

MSC therapy enhances lung repair following VILI via a paracrine mechanism that may be keratinocyte growth factor-dependent.

<em>Keratinocyte</em> apoptosis is a central element in the regulation of hair follicle regression (catagen), yet the exact location and the control of follicular <em>keratinocyte</em> apoptosis remain obscure. To generate an "apoptomap" of the hair follicle, we have studied selected apoptosis-associated parameters in the C57BL/6 mouse model for hair research during normal and pharmacologically manipulated, pathological catagen development. As assessed by terminal deoxynucleotide transferase dUTP fluorescein nick end-labeling (TUNEL) stain, apoptotic cells not only appeared in the regressing proximal follicle epithelium but, surprisingly, were also seen in the central inner root sheath, in the bulge/isthmus region, and in the secondary germ, but never in the dermal papilla. These apoptosis hot spots during catagen development correlated largely with a down-regulation of the Bcl-<em>2</em>/Bax ratio but only poorly with the expression patterns of interleukin-1beta converting enzyme, p55TNFR, and Fas/Apo-1 immunoreactivity. Instead, a higher correlation was found with p75NTR expression. During cyclophosphamide-induced follicle dystrophy and alopecia, massive <em>keratinocyte</em> apoptosis occurred in the entire proximal hair bulb, except in the dermal papilla, despite a strong up-regulation of Bax and p75NTR immunoreactivity. Selected receptors of the tumor necrosis <em>factor</em>/nerve <em>growth</em> <em>factor</em> family and members of the Bcl-<em>2</em> family may also play a key role in the control of follicular <em>keratinocyte</em> apoptosis in situ.

Proper regulation of <em>keratinocyte</em> differentiation within the epidermis and follicular epithelium is essential for maintenance of epidermal barrier function and hair <em>growth</em>. The signaling intermediates that regulate the morphological and genetic changes associated with epidermal and follicular differentiation remain poorly understood. We tested the hypothesis that reactive oxygen species (ROS) generated by mitochondria are an important regulator of epidermal differentiation by generating mice with a <em>keratinocyte</em>-specific deficiency in mitochondrial transcription <em>factor</em> A (TFAM), which is required for the transcription of mitochondrial genes encoding electron transport chain subunits. Ablation of TFAM in <em>keratinocytes</em> impaired epidermal differentiation and hair follicle <em>growth</em> and resulted in death <em>2</em> weeks after birth. TFAM-deficient <em>keratinocytes</em> failed to generate mitochondria-derived ROS, a deficiency that prevented the transmission of Notch and β-catenin signals essential for epidermal differentiation and hair follicle development, respectively. In vitro <em>keratinocyte</em> differentiation was inhibited in the presence of antioxidants, and the decreased differentiation marker abundance in TFAM-deficient <em>keratinocytes</em> was partly rescued by application of exogenous hydrogen peroxide. These findings indicate that mitochondria-generated ROS are critical mediators of cellular differentiation and tissue morphogenesis.

A plethora of <em>growth</em> <em>factors</em> regulate <em>keratinocyte</em> proliferation and differentiation that control hair morphogenesis and skin barrier formation. Wavy hair phenotypes in mice result from naturally occurring loss-of-function mutations in the genes for TGF-alpha and EGFR. Conversely, excessive activities of TGF-alpha/EGFR result in hairless phenotypes and skin cancers. Unexpectedly, we found that mice lacking the Trpv3 gene also exhibit wavy hair coat and curly whiskers. Here we show that <em>keratinocyte</em> TRPV3, a member of the transient receptor potential (TRP) family of Ca(<em>2</em>+)-permeant channels, forms a signaling complex with TGF-alpha/EGFR. Activation of EGFR leads to increased TRPV3 channel activity, which in turn stimulates TGF-alpha release. TRPV3 is also required for the formation of the skin barrier by regulating the activities of transglutaminases, a family of Ca(<em>2</em>+)-dependent crosslinking enzymes essential for <em>keratinocyte</em> cornification. Our results show that a TRP channel plays a role in regulating <em>growth</em> <em>factor</em> signaling by direct complex formation.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) administered as a single intratracheal injection causes a prominent dose-dependent proliferation of type II alveolar epithelial cells in the lungs of adult rats. The increase in mitotically active alveolar cells histologically appears as a micropapillary epithelial cell hyperplasia after <em>2</em> d and peaks after 3 d in the form of monolayers of cuboidal epithelial cells lining alveolar septae. Proliferating cell nuclear antigen immunohistochemistry confirmed the profound proliferative response induced by KGF. The hyperplastic alveolar lining cells contain immunoreactive surfactant protein B and are ultrastructurally noted to contain lamellar inclusions characteristic of surfactant-producing type II pneumocytes. Mild focal bronchiolar epithelial hyperplasia is noted but is much less striking than the proliferation of type II pneumocytes. Large airways are unaffected by KGF. Daily intravenous injection of KGF is also able to cause pneumocyte proliferation. The normal adult rat lung constitutively expresses both KGF and KGF receptor mRNA, suggesting that endogenous KGF may be implicated in the paracrine regulation of the <em>growth</em> of pneumocytes. In conclusion, KGF rapidly and specifically induces proliferation and differentiation of type II pneumocytes in the normal adult lung.

The transplantation of well defined populations of precursor cells offers a means of repairing damaged tissue and of delivering therapeutic compounds to sites of injury or degeneration. For example, a functional immune system can be reconstituted by transplantation of purified haematopoietic stem cells, and transplanted skeletal myoblasts and <em>keratinocytes</em> can participate in the formation of normal tissue in host animals. Cell transplantation in the central nervous system (CNS) has been proposed as a means of correcting neuronal dysfunction in diseases associated with neuronal loss; it might also rectify glial cell dysfunction, with transplanted oligodendrocyte precursor cells eventually allowing repair of demyelinating damage in the CNS. Here we use co-operating <em>growth</em> <em>factors</em> to expand purified populations of oligodendrocyte type-<em>2</em> astrocyte (O-<em>2</em>A) progenitor cells for several weeks in vitro. When injected into demyelinating lesions in spinal cords of adult rats, created in such a way as to preclude host-mediated remyelination, these expanded populations are capable of producing extensive remyelination. In addition, transplantation of O-<em>2</em>A progenitor cells genetically modified to express the bacterial beta-galactosidase gene gives rise to beta-galactosidase-positive oligodendrocytes which remyelinate demyelinated axons within the lesion. These results offer a viable strategy for the manipulation of neural precursor cells which is compatible with attempts to repair damaged CNS tissue by precursor transplantation.

To assess tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and clinical activity of the dual epidermal <em>growth</em> <em>factor</em> receptor (EGFR) 1 and <em>2</em> (HER<em>2</em>) tyrosine kinase inhibitor BIBW <em>2</em>99<em>2</em>. An escalating schedule of once-daily (OD) BIBW <em>2</em>99<em>2</em> for 14 days followed by 14 days off medication was explored. Thirty-eight patients were enrolled. Dose levels were 10, <em>2</em>0, 30, 45, 70, 85, and 100 mg. At 100 mg dose-limiting toxicity (DLT) (common toxicity criteria grade 3 skin rash and grade 3 diarrhoea despite treatment with loperamide) occurred in two patients. In the next-lower dose of 70 mg, DLT (grade 3 fatigue and ALAT elevation) occurred in one of six patients. An intermediate dose level of 85 mg was studied. Here DLT occurred in two patients (grade 3 diarrhoea despite treatment and grade <em>2</em> diarrhoea lasting more than 7 days despite treatment). An additional 1<em>2</em> patients were treated at 70 mg. BIBW <em>2</em>99<em>2</em> PK after single and multiple doses revealed moderately fast absorption, and no deviation from dose proportionality. Pharmacodynamics analysis in skin biopsies did not show significant changes in EGFR-associated biomarkers. However, a significant inhibitory effect on the proliferation index of epidermal <em>keratinocytes</em> was observed. No partial or complete responses were observed, stable disease lasting more than four cycles was seen in seven patients. The recommended dose for studies with BIBW <em>2</em>99<em>2</em> for 14 days followed by 14 days off medication is 70 mg OD.

Epidermal <em>growth</em> <em>factor</em> receptor (EGFR) is a key regulator of <em>keratinocyte</em> biology. However, the physiological role of EGFR in vivo has not been well established. To analyze the role of EGFR in skin, we have generated transgenic mice expressing an EGFR dominant negative mutant in the basal layer of epidermis and outer root sheath of hair follicles. Mice expressing the mutant receptor display short and waved pelage hair and curly whiskers during the first weeks of age, but subsequently pelage and vibrissa hairs become progressively sparser and atrophic. Eventually, most mice present severe alopecia. Histological examination of the skin of transgenic mice shows striking alterations in the development of hair follicles, which fail to enter into catagen stage. These alterations eventually lead to necrosis and disappearance of the follicles, accompanied by strong infiltration of the skin with inflammatory elements. The interfollicular epidermis of these mice shows marked hyperplasia, expression of hyperproliferation-associated keratin K6 and increased 5-bromo-<em>2</em>-deoxyuridine incorporation. EGFR function was inhibited in transgenic skin <em>keratinocytes</em>, since in vivo and in vitro autophosphorylation of EGFR was almost completely abolished on EGF stimulation. These results implicate EGFR in the control of hair cycle progression, and provide new information about its role in epidermal <em>growth</em> and differentiation.

The immortal human <em>keratinocyte</em> line HaCaT is frequently used as a paradigm for skin <em>keratinocytes</em> in vitro because of its highly preserved differentiation capacity. HaCaT cells form a nearly regular epidermal architecture when transplanted onto subcutaneous tissue of athymic mice. In order to analyze further their differentiation capacity in vitro, HaCaT cells were studied in organotypic cocultures on top of collagen gels containing human dermal fibroblasts. Within 1 wk HaCaT cells formed a still dysplastic epithelium, the thickness of which correlated with the number of fibroblasts in the collagen gel. With further culture time of up to 3 wk a remarkably well structured and differentiated squamous epithelium developed. After 1 wk, keratins 10 and 16, involucrin, and transglutaminase I were expressed in suprabasal layers, whereas filaggrin, keratin <em>2</em>e, and loricrin appeared after <em>2</em>-3 wk. Within this time, a nearly complete basement membrane had formed including hemidesmosomes and anchoring fibrils. Epithelial cell proliferation became restricted to the basal layer after <em>2</em> and 3 wk. Using the TdT-mediated dUTP nick end labeling assay, fragmentation of DNA was detectable in nuclei of the parakeratotic stratum corneum. Ultrastructurally, many features of keratinization accumulated after <em>2</em> and 3 wk, though an orthokeratotic keratinization was not achieved, in contrast to HaCaT transplants. This differentiation deficiency - as compared with normal <em>keratinocytes</em> -- might be due to a lack of paracrine <em>factors</em> important for <em>keratinocyte</em> differentiation or to a reduced sensitivity of these cells. Nevertheless, this high degree of differentiation under organotypic conditions qualifies this cell line as an appropriate model for elucidation of the molecular mechanisms regulating <em>keratinocyte</em> <em>growth</em> and differentiation and for use in pharmacotoxicology.

Signals transmitted from mesenchyme to epithelia or vice versa constitute the basis of reciprocal epithelial-mesenchymal interactions. As a first step toward understanding epithelial-mesenchymal interactions on the ocular surface where the transit amplifying cell-containing corneal epithelium is anatomically separated from the stem cell-containing limbal epithelium, we sought to characterize the expression patterns of cytokines and their receptors by primary epithelial and early-passaged fibroblast cultures of human cornea and limbus. Northern hybridization with oligonucleotide and cDNA probes to a total of <em>2</em>5 cytokines and 1<em>2</em> of their receptors revealed that the positively expressed cytokines could be divided into the following four patterns. Type I: TGF-alpha, IL-1 beta, and PDGF-B were expressed exclusively by epithelial cells but their respective receptors EGFR and IL-1R were predominantly and PDGFR-beta was exclusively expressed by fibroblasts. Type II: IGF-I, TGF-beta 1, -beta <em>2</em>, LIF, and bFGF, and their receptors were expressed by both epithelial cells and fibroblasts. FGFR-1 (flg) and FGFR-<em>2</em> (bek) were expressed more by fibroblasts and bFGF was expressed more by corneal than limbal epithelial cells. Type III: <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) were expressed exclusively by fibroblasts and their respective receptors, KGFR and c-met, were predominantly expressed by epithelial cells. Combined with RT-PCR, the quantity of KGF and KGFR transcripts was highest in limbal fibroblasts and epithelial cells, respectively. In contrast, the quantity of HGF and HGFR (c-met) transcripts was highest in corneal fibroblasts and epithelial cells, respectively. Type IV: M-CSF and IL-8 were expressed by fibroblasts and/or epithelial cells but their receptors were not expressed by epithelial cells nor fibroblasts, but by immune or inflammatory cells. In addition to these potential paracrine actions, autocrine actions mediated by TGF-alpha/EGFR, IL-1 beta/IL1-R, and bFGF/FGFR-1 were more expressed by corneal than limbal epithelial cells. Immunofluorescence staining on human corneoscleral cryosections confirmed that EGFR and bFGF were not expressed by the limbal basal epithelium, but expressed strongly by the corneal epithelium, a pattern consistent with Northern hybridization. These results indicate that ocular surface epithelial cells and fibroblasts can express a myriad of cytokines, among which the first three patterns constitute the network of potential epithelial-mesenchymal cytokine dialogues. The difference of certain cytokine expression between corneal and limbal regions suggests that this network participates in normal epithelial <em>growth</em> and differentiation, and plays an important role in wound healing.

Southern blot-hybridization analysis of DNAs from human tumors demonstrated amplification of the epidermal <em>growth</em> <em>factor</em> (EGF) receptor gene in 10 of 1<em>2</em> squamous cell carcinoma cell lines tested and in none of 18 tumor cell lines of nonsquamous cell carcinomas. The degree of amplification in the squamous cells varied from <em>2</em>- to 50-fold relative to the epidermal <em>keratinocyte</em>. Hybridization analysis of the RNA showed that the amplification of the EGF receptor gene is accompanied with an increase of the 5.6 kilobases of EGF receptor mRNA. Scatchard plot analysis and sodium dodecyl sulfate-polyacrylamide gel analysis of the EGF receptor revealed that the synthesis of the EGF receptor is also greater in the cells with amplified EGF receptor gene. In contrast, Southern blot analysis of DNAs of primary tumors showed that incidence of amplification of the EGF receptor gene in squamous cells (1 of 6) was almost as frequent as in nonsquamous cells (1 of 4). These results show that amplification of the EGF receptor gene is commonly found in various tumors. In addition, our data suggest that primary squamous cell carcinomas with amplified EGF receptor gene may readily adapt to <em>growth</em> in tissue culture.

BACKGROUND

During wound healing, <em>keratinocytes</em> detach from the basement membrane and migrate to cover the exposed connective tissue. Subsequently, the wound clot is degraded gradually and replaced by the epithelial cells and the granulation tissue. Both of these processes are likely to be affected by matrix-modifying enzymes. Type IV collagenases are members of the matrix metalloproteinase family (MMP), which are known to degrade several matrix components. The aim of this study was to investigate the expression of MMP-<em>2</em> and MMP-9 (7<em>2</em>-kd and 9<em>2</em>-kd type IV collagenases, respectively) during early human wound healing.

METHODS

Experimental wounds were created in human oral mucosa and biopsies were taken 1, 3, and 7 days after wounding. In situ hybridization on paraffin sections was used for the detection of messenger RNAs coding for MMP-<em>2</em> and MMP-9, and the secretion of MMPs into the oral cavity after wounding was followed by zymography. Regulation of MMP-<em>2</em> and MMP-9 expression by cytokines was studied using cultured mucosal <em>keratinocytes</em>, gingival fibroblasts, and wound granulation tissue fibroblasts.

RESULTS

By in situ hybridization, the expression of MMP-<em>2</em> was localized in the connective tissue fibroblasts and endothelial cells during all phases of wound healing. Mucosal epithelium was practically negative for MMP-<em>2</em> expression. MMP-9 messenger RNA was found in mucosal epithelium on days 1, 3, and 7. A strong signal was localized in basal and suprabasal cell layers in the nonwounded area, while only the basal cell layer was MMP-9 positive in the migrating epithelial sheet. Seven days after wounding, granulation tissue exhibited an unusually strong signal for MMP-9 messenger RNA. Wound fluid contained mainly MMP-9, the amount of which was highest in two- to four-day-old secretions. None of the cytokines tested (transforming growth factor beta-1, interleukin-1 beta, basic fibroblast growth factor, tumor necrosis factor-alpha, interferon-gamma) were able to regulate MMP-<em>2</em> expression in cultured wound fibroblasts. However, keratinocyte MMP-9 production was enhanced by interleukin-1 beta, transforming growth factor beta-1, and tumor necrosis factor-alpha.

CONCLUSIONS

During wound healing, MMP-9 is suggested to be involved in keratinocyte migration and granulation tissue remodelling. Expression of MMP-<em>2</em> remains stable during wound healing.