Beyond Reproduction: Pituitary Hormone Actions on Bone.

The long-held belief that pituitary hormones act solely on master targets was first questioned when we documented G protein-coupled receptors for thyroid-stimulating hormone, follicle-stimulating hormone, adrenocorticotrophic hormone, oxytocin, and vasopressin on bone cells. These evolutionarily conserved hormones and their receptors are known to have primitive roles, and exist in invertebrate species as far down as coelenterates. It is not surprising therefore that each such hormone has multiple hitherto unrecognized functions in mammalian integrative physiology, and hence, becomes a potential target for therapeutic intervention. Here we discuss the skeletal actions of pituitary hormones.

The oxytocin-bone axis.

We recently demonstrated a direct action of oxytocin (OT) on skeletal homeostasis, mainly mediated through stimulation of osteoblasts (OBs) formation and through the reciprocal modulation of osteoclast (OCs) formation and function. Thus, mice lacking the hormone or its receptor develop a low turnover osteoporosis that worsens with age in both sexes. The skeletons of OT (Ot) and OT receptor (Oxtr) null mice display a pronounced decrease in vertebral and femoral trabecular volume. At the cellular level, OBs from Ot KO and Oxtr KO mice exhibit lower mineralization activity and, at the mRNA level, all master genes for osteoblast differentiation are down-regulated. Moreover, OT has dual effects on OCs: it increases osteoclast formation both directly, by activating nuclear factor kB (NFkB) and mitogen-activated protein kinase (MAPK) signalling and, indirectly, through the up-regulation of receptor activator nuclear factor-kappaB ligand synthesis by OBs. On the other hand, it inhibits bone resorption by triggering cytosolic Ca(2+) release and nitric oxide synthesis in mature OCs. OT is locally produced by osteoblasts acting as paracrine-autocrine regulators of bone formation modulated by oestrogens. The oestrogen signal involved in this feedforward circuit is nongenomic because it requires an intact MAPK kinase signal transduction pathway, instead of the classical nuclear translocation of oestrogen receptor. The ability of oestrogen to increase bone mass in vivo is to some extent OXTR-dependent. Thus, Oxtr KO mice injected 17ß-oestradiol did not show any effects on bone formation parameters, whereas the same treatment increases trabecular and cortical bone in wild-type mice. An intact OT autocrine-paracrine circuit appears to be essential for optimal skeletal remodelling.

Myeloma cells suppress osteoblasts through sclerostin secretion.

Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of ß-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

Soluble decoy receptor 3 modulates the survival and formation of osteoclasts from multiple myeloma bone disease patients.

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor (TNF) receptor superfamily, is known to be involved in cell survival and osteoclast (OC) formation. In this study, we show that malignant plasma cells and T lymphocytes from multiple myeloma (MM) bone disease patients, as well as Karpas 909, a human myeloma cell line, directly produce DcR3. By interacting with FasL, this molecule could inhibit OC apoptosis. In fact, the use of a neutralizing anti-DcR3 antibody induces a reduction of cell viability with a consequent increase of apoptotic cell number, the activation of caspase-8 and -3, and DNA fragmentation. Furthermore, we show that DcR3 supports OC formation in samples from MM patients through the upregulation of RANKL and TNFalpha by T lymphocytes and only TNFalpha by CD14+ cells. In conclusion, our data provide the first evidence of the expression of DcR3 in MM, and the involvement of this molecule in supporting the survival and formation of OCs from MM bone disease patients. The production of DcR3 by T lymphocytes confers these cells a role in the pathogenesis of bone disease associated with MM.

L: -carnitine fumarate and isovaleryl-L: -carnitine fumarate accelerate the recovery of bone volume/total volume ratio after experimetally induced osteoporosis in pregnant mice.

Anabolic skeletal agents have recently broadened the therapeutic options for osteoporosis by directly stimulating bone formation and improving bone turnover, bone density, bone size, and bone microarchitecture. We recently demonstrated that two new L: -carnitine derivatives, L: -carnitine fumarate (LC) and isovaleryl-L: -carnitine fumarate (Iso-V-LC), stimulated osteoblast proliferation and differentiation. We here investigated, by histomorphometry in a mouse model of osteoporosis, the impact of these compounds on the repair of trabecular bone and the osteoblast involvement in this process. Fifty-nine inbred adult female CD1 mice in pregnancy were assigned to four treatment groups: (1) controls, mice fed a standard normocalcemic pre- and postpartal diet; (2) Hypo, mice fed a low-calcium isocaloric prepartal diet and a standard postpartal diet; (3) LC, mice fed a group 2-type diet supplemented post-partum with LC; (4) Iso-V-LC, mice fed a group 2-type diet supplemented post-partum with Iso-V-LC. Bone volume/total volume ratio (BV/TV), bone perimeter, osteoblast surface/bone surface, and osteoblast number/bone surface were measured from sections of L3 and L4 vertebral bodies obtained from animals killed on the day of delivery (controls and Hypo) and on days 7, 14, and 21 after delivery (all groups). BV/TV and all osteoblast-based indexes were significantly higher in LC and Iso-V-LC than in Hypo mice at each time point, and Iso-V-LC at the end of the treatment attained levels observed in controls. In conclusion, Iso-V-LC and, to a lesser extent, LC accelerated the recovery of normal BV/TV level after a hypocalcemic diet.

Role of tumour necrosis factor alpha, but not of cyclo-oxygenase-2-derived eicosanoids, on functional and morphological indices of dystrophic progression in mdx mice: a pharmacological approach.

The role of tumour necrosis factor (TNF)-alpha or cyclo-oxygenase-2 (COX-2) eicosanoids in dystrophinopathies has been evaluated by chronically treating (4-8 weeks) adult dystrophic mdx mice with the anti-TNF-alpha etanercept (0.5 mg/kg) or the COX-2 inhibitor meloxicam (0.2 mg/kg). Throughout the treatment period the mdx mice underwent a protocol of exercise on treadmill in order to worsen the pathology progression; gastrocnemious muscles from exercised mdx mice showed an intense staining for TNF-alpha by immunohistochemistry. In vivo, etanercept, but not meloxicam, contrasted the exercise-induced forelimb force drop. Electrophysiological recordings ex vivo, showed that etanercept counteracted the decrease in chloride channel function (gCl), a functional index of myofibre damage, in both diaphragm and extensor digitorum longus (EDL) muscle, meloxicam being effective only in EDL muscle. None of the drugs ameliorated calcium homeostasis detected by electrophysiology and/or spectrofluorimetry. Etanercept, more than meloxicam, effectively reduced plasma creatine kinase (CK). Etanercept-treated muscles showed a reduction of connective tissue area and of pro-fibrotic cytokine TGF-beta1 vs. untreated ones; however, the histological profile was weakly ameliorated. In order to better evaluate the impact of etanercept treatment on histology, a 4-week treatment was performed on 2-week-old mdx mice, so to match the first spontaneous degeneration cycle. The histology profile of gastrocnemious was significantly improved with a reduction of degenerating area; however, CK levels were only slightly lower. The present results support a key role of TNF-alpha, but not of COX-2 products, in different phases of dystrophic progression. Anti-TNF-alpha drugs may be useful in combined therapies for Duchenne patients.

T cells support osteoclastogenesis in an in vitro model derived from human periodontitis patients.

BACKGROUND: Periodontitis is characterized by alveolar bone destruction; however, the mechanisms responsible for bone damage are poorly understood. It has been reported that T cells are implicated in the pathogenesis of periodontitis. It has been also demonstrated that activated T lymphocytes secrete receptor activator of nuclear factor-kappa B ligand (RANKL) and can support the differentiation of monocytes into resorbing osteoclasts (OCs). Therefore, the purpose of this study was to examine the OC formation in periodontitis patients (PP) and the role of T cells in osteoclastogenesis. METHODS: To study OC formation, we used an in vitro model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from PP and controls. In parallel, T-cell-depleted PBMCs from the same patients were also established. The expression of RANKL and tumor necrosis factor-alpha (TNF-alpha) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot in fresh T cells isolated from PP and controls. Functional antibodies, anti-RANKL and anti-TNF-alpha, were utilized to study osteoclastogenesis in PBMC cultures from PP. RESULTS: We showed that, in unfractionated PBMCs from PP, the OCs spontaneously developed in a T-cell-dependent way. The addition of macrophage colony stimulating factor (MCSF) and RANKL was necessary to promote the osteoclastogenesis in T-cell-depleted PBMC cultures from PP and in unfractionated PBMCs from periodontally healthy controls. Moreover, freshly isolated T cells from PBMCs of PP overexpressed RANKL and TNF-alpha. Finally, functional anti-RANKL and anti-TNF-alpha antibodies significantly inhibited osteoclastogenesis. CONCLUSION: Our data suggest that T cells support spontaneous osteoclastogenesis in PP via RANKL and TNF-alpha overexpression.

L-carnitine and isovaleryl L-carnitine fumarate positively affect human osteoblast proliferation and differentiation in vitro.

Age-related bone loss is characterized by decreased osteoblast activity, possibly related to the reduction of energy production. Carnitine promotes energy availability and its concentration declines with age; Therefore, two Carnitine derivatives, L-carnitine fumarate (LC) and isovaleryl L-carnitine fumarate (Iso-V-LC), have been tested on several parameters of human osteoblasts in vitro. Both compounds significantly increased osteoblast activity, but the new compound Iso-V-LC was more efficient than LC at lower concentrations. They both significantly enhanced cell proliferation, [3H]-proline incorporation and the expression of collagen type I (COLLI), and the bone sialoproteins (BSPs) and osteopontin (OPN). The percentage of alkaline phosphatase (ALP)-positive cells and the secretion of osteocalcin were not modified by LC and Iso-V-LC. Both molecules increased the formation of mineralized nodules, but Iso-V-LC reached the maximum effect at a concentration 10-fold lower than that of LC. Furthermore, we showed that insulin-like growth factor (IGF)-I and IGF-II mRNA levels were not modified by the treatment. However, the two compounds induced an increase of insulin-like growth factor binding protein (IGFBP)-3 and a decrease of IGFBP-5 in both osteoblast lysates and the extracellular matrix (ECM). In conclusion these data suggest that carnitine and, in particular, its new derivative, Iso-V-LC supplementation in the elderly may stimulate osteoblast activity and decrease age-related bone loss.

HGF and M-CSF modulate adhesion of MDA-231 breast cancer cell by increasing osteopontin secretion.

Recent studies reported an increased expression of osteopontin (OPN) in metastatic breast cancer cells, but the mechanisms modulating OPN production and the interaction of the cells with the secreted protein are far from clear. In this work, we utilized as an experimental system the cell line MDA-231 and we showed that HGF and M-CSF significantly enhance their adhesion onto OPN. Furthermore, in the presence of HGF and M-CSF, MDA-231 cells can adhere when plated onto BSA via increased OPN secretion. Moreover HGF and M-CSF induce de novo synthesis of OPN. In conclusion, these data suggest that HGF and M-CSF stimulate OPN production by MDA-231 cells, and that OPN is subsequently used as a substrate for cell adhesion.

Rat hindlimb unloading by tail suspension reduces osteoblast differentiation, induces IL-6 secretion, and increases bone resorption in ex vivo cultures.

In this research we utilized tail-suspended rats as an in vivo model for bone loss studies in order to investigate the effects of the tail suspension on the structure of the suspended bones and in ex vivo cultures the activities of trabecular osteoblasts, marrow-derived osteogenic cells, and osteoclasts obtained from treated animals, compared with untreated controls. After a 5-day hind limb unloading, trabecular thinning was already evidenced in the tibial primary spongiosa. In the secondary spongiosa, the bone formation activity was reduced whereas osteoclastic parameters were not yet altered. Bone marrow-derived osteogenic cells and differentiated osteoblasts from enzymatic digestion of posterior limb trabecular bone were prepared from 5 day tail-suspended rats and from normally loaded rats as controls. Cell morphology, alkaline phosphatase (ALPH) activity, production of mineral matrix, osteocalcin, and IL-6 secretion were evaluated in both cell populations. Tail suspension reduced the osteogenic potential of stromal marrow cells and of already differentiated osteoblasts. In fact, ALP positive colonies were significantly reduced in number and were smaller in size compared with controls and bone nodules formed in permissive conditions were also significantly fewer and smaller, whereas in cultures of cells from control conditions, large mineralizing nodules were formed. Osteocalcin secretion was not affected by unloading. Finally, IL-6 concentration was increased in marrow-derived cells from treated rats compared with controls. Primary cultures of osteoclasts were obtained from the nonadherent fraction of the bone marrow of the same animals. The number of TRAP positive cells in culture from tail-suspended rats was significantly increased, as well as bone resorption activity, measured as resorbed surfaces of a suitable synthetic hydroxyapatite, compared with controls. These data clearly suggest that skeletal unloading not only reduces the osteogenic potential of osteoblastic cells but induces an increased osteoclastogenesis and osteoclast activity in ex vivo cultures. They also indicate for the first time that a possible mediator responsible for the increased osteoclastogenesis could be represented by the IL-6 whose secretion by bone marrow cells was significantly enhanced by unloading.

Osteobiology, strain, and microgravity. Part II: studies at the tissue level.

Loading microgravity, and/or defective mechanical strain-forces have important effects on bone cells and bone quality and quantity. The complex mechanisms induced by strain and microgravity on bone cells have been reviewed in Part I of this paper. In Part II, we have considered the data on the alterations induced by unloading and microgravity on the skeleton and the mechanisms that are involved at the tissue level in animals and humans.

Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts.

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage.

Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERalpha), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERalpha mRNA, together with immunocytochemical analysis using a human ERalpha-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERalpha in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERalpha expression was detected in human bone marrow cultures treated with 1,25(OH)2D3 and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)2D3. However, in an in vitro model of human osteoclast formation, no ERalpha expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERalpha in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERalpha, but osteoclast maturation and bone resorption is associated with loss of ERalpha expression. This indicates that ERalpha expression and regulation may play a role in osteoclast formation.

Alendronate reduces adhesion of human osteoclast-like cells to bone and bone protein-coated surfaces.

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40-45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as alphaVbeta3 integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration.

Activation of alphav beta3 integrin on human osteoclast-like cells stimulates adhesion and migration in response to osteopontin.

Integrins mediate cell adhesion and can induce different cellular responses, including changes in intracellular pH, changes and oscillation in intracellular free calcium, and protein phosphorylation on tyrosine. During bone resorption, the integrin alphav beta3 regulates adhesion of osteoclasts to bone extracellular matrix proteins, such us osteopontin (Opn). Adhesion via alphav beta3 is followed by osteoclast polarization onto the bone surface and by the onset of bone resorption. To characterize these events at the molecular level, we investigated the state of activation of alphav beta3 on the human osteoclast-like cell line GCT23 using the monoclonal antibody AP5 which binds to and can induce, under low calcium conditions, activated alphav beta3. By flow cytometry, approximately 50% of alphav beta3 on the surface of the osteoclast-like cell line GCT23 was reactive with AP5 and was therefore in the activated state. Incubation with AP5 in the presence of low calcium concentrations increased activated alphav beta3 to 90-100%. Activation of alphav beta3 increased the efficiency of GCT23 adhesion to Opn by 2-fold. Furthermore, haptotactic migration on Opn was also enhanced about 40% compared to control. We propose that changes in the activation state of alphav beta3 may be a regulation point for osteoclasts during bone resorption.

Retinoic acid induces cell proliferation and modulates gelatinases activity in human osteoclast-like cell lines.

The effect of Retinoic Acid (RA) on human osteoclast-like cell lines, obtained from Giant Cell tumors (GCT) of bone, has been investigated evaluating its action on bone resorption, cell proliferation, microtubular organization and gelatinases expression and activity. Increasing concentrations of RA significantly dose-dependently decreased GCTs bone resorption, while 10(-7) M RA promoted an increase of cell proliferation. By immunofluorescence we demonstrated that GCTs express A and B gelatinases and, by zymography, that their activity was enhanced in medium collected from GCTs cultured in the presence of 10(-7) M RA. These data indicate that RA increases cell proliferation and modulates metalloproteinases (MMPs) activity, crucial events during the migration of osteoclast precursors toward bone surfaces.

Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro.

Hepatocyte growth factor (HGF), also known as scatter factor, is a powerful motogen, mitogen, and morphogen produced by cells of mesodermal origin, acting on epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. We show that the HGF receptor is expressed by human primary osteoclasts, by osteoclast-like cell lines, and by osteoblasts. In both cell lineages, HGF stimulation triggers the receptor kinase activity and autophosphorylation. In osteoclasts, HGF receptor activation is followed by increase in intracellular Ca2+ concentration and by activation of the pp60c-Src kinase. HGF induces changes in osteoclast shape and stimulates chemotactic migration and DNA replication. Osteoblasts respond to HGF by entering the cell cycle, as indicated by stimulation of DNA synthesis. Interestingly, osteoclasts were found to synthesize and secrete biologically active HGF. These data strongly suggest the possibility of an autocrine regulation of the osteoclast by HGF and a paracrine regulation of the osteoblast by the HGF produced by the osteoclast.

Human osteoclast-like cells selectively recognize laminin isoforms, an event that induces migration and activates Ca2+ mediated signals.

Osteoclast precursors are chemotactically attracted to sites of bone resorption via migration pathways that include transendothelial crossing in blood capillaries. Transendothelial migration involves poorly understood interactions with basal lamina molecules, including laminins. To investigate osteoclast-laminin interactions, we used human osteoclast-like cell lines obtained from giant cell tumors of bone (GCT 23 and GCT 24). These cell lines are a well-characterized model for osteoclast functions, such as bone resorption and the behaviour of osteoclast precursors. Both GCT cell lines adhered to laminin-2 (merosin) coated wells in standard adhesion assays, but failed to adhere to laminin-1 (EHS-laminin). By light microscopy, GCT cells on laminin-2 were partially spread, with a motile morphology. None of the anti-integrin antibodies tested inhibited GCT cells adhesion to laminin-2. Peptides containing the integrin adhesion site RGD or the laminin adhesion sequence IKVAV did not inhibit GCT cell adhesion to laminin-2. By immunofluorescence, beta 1 integrins were organized in focal adhesions. However, in the presence of monensin this reorganization of beta 1 integrins was abolished, indicating that it was probably due to secretion of fibronectin by GCT cells subsequent to adhesion to laminin-2. GCT cells transmigrated through membranes coated with laminin-2, much more efficiently than through membranes coated with collagen. Migration was induced by osteocalcin, as a chemoattractant, in a dose-dependent manner. At low osteocalcin concentrations, transmigration was detectable on laminin-2 but not collagen. In cells loaded with fura-2, a sharp increase in intracellular Ca2+ was detected upon addition of soluble laminin-2, but not laminin-1, due to release from thapsigargin-dependent intracellular stores. In summary, osteoclasts may recognize laminin isoforms differentially. Initial adhesion to laminin-2 appears to be due to integrin-independent mechanisms. Such adhesion, though, may trigger secretion of fibronectin that could then support spreading and efficient chemotactic migration. These mechanisms may play an important role in facilitating chemotactic migration of osteoclast precursors toward the bone surface.

Altered expression of basement membrane proteins and their integrin receptors in lichen planus: possible pathogenetic role of gelatinases A and B.

Lichenoid lesions of the skin are characterized by a band-like dermal inflammatory infiltrate and structural alterations of the basement membrane (BM). The etiopathogenesis of these lesions, of which lichen planus (LP) is perhaps the prototypic example, is unknown. Acute cases of LP are accompanied by the destruction of epidermal BM, degeneration of basal keratinocytes with loss of tonofilaments and hemidesmosomes, vesicular alterations, and even blister formation. Chronic LP is characterized by hyperkeratosis and acanthosis in the epidermis, fibrosis, and dense infiltrate in dermis. We found that acute LP lesions are characterized by uneven or absent immunostaining for laminin-5, laminin-1, and collagen type IV. Distribution and activity of gelatinases matrix metalloproteinase (MMP)-9 and MMP-2, and a specific inhibitor of MMP-2, tissue inhibitor of metalloprotein-2, were analyzed. The expression and activity of MMP-2 were increased, whereas tissue inhibitor of metalloprotein-2 expression was weak in the involved areas during the acute phase of LP. Moreover, we demonstrated in vitro that MMP-2 is directly capable of digesting laminin-5 gamma 2 chains to yield a 80-kd fragment. We also observed a weak or absent staining of all examined integrin receptors in the acute LP lesions. In chronic lesions, the staining of BM components was similar to normal controls. In these sections, normal expression of gelatinases and inhibitor was observed. There was, however, up-regulation and altered polarity of integrin receptors. These results indicate a link between overexpression of gelatinases, BM disruption, and altered integrin expression. In LP, the digestion of BM by MMP-2 may contribute to the pathogenesis by inducing altered integrin expression in basal keratinocytes and ultimately blister formation.