ARTICLE |
Correspondence to: Aarne Oikarinen, Dept. of Dermatology, Univ. of Oulu, Fin-90220 Oulu, Finland.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined the expression of laminin-5 and its integrin receptors during reepithelialization of human wounds. We used suction blisters of skin as a model of keratinocyte migration on a basement membrane matrix and mucosal full-thickness wounds as a model in which keratinocytes migrate in a provisional matrix. An animal model, in which human epidermal keratinocytes were injected into the back of athymic mice, was used to follow the deposition of the basement membrane components. In 4-day-old blisters, about 2050 cells at the leading edge of the migrating tongue showed cytoplasmic laminin-5 immunostaining. Laminin-5 mRNA was detected in 1530 cells at the leading edge of the migrating epidermis. 3ß1 and
6ß4 integrins were found in membrane projections of the migrating basal cells and also in suprabasal cell layers, suggesting their combined role in binding laminin-5. In mucosal wounds, laminin-5 was the only basement membrane zone component that was deposited between the clot and the migrating keratinocytes. In the animal model, linear deposition of laminin-5 and
6ß4 integrin was already seen on Day 2, whereas the other basement membrane zone components were not yet organized. The results suggest that, regardless of the injury and the microenvironment, laminin-5 plays an essential role in the interaction between wound keratinocytes and the surrounding matrix. (J Histochem Cytochem 46:353360, 1998)
Key Words: wound healing, suction blister, anchoring filament, integrins, cytokines, in situ hybridization
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Laminin-5 (LM-5), also known as kalinin/nicein/epiligrin, is a recently characterized component of anchoring filaments localized in the basement membrane (BM) region of the skin, mucosa, lung, and intestine (3, ß3, and
2 (
LM-5 appears to be one of the best ligands for keratinocyte adhesion and migration. It can also promote the formation of hemidesmosomes in vitro (3ß1 and
6ß4 integrins can act as cell receptors for LM-5 in keratinocytes (
The aim of the present investigation was to study the role of LM-5 in physiological keratinocyte migration regardless of the microenvironment and injury type of the wound. We used three different experimental models: a suction blister of skin, in which keratinocytes migrate on a BM substratum; a full-thickness mucosal wound, in which keratinocytes migrate through the clot matrix; and an animal model, in which early BM organization can be followed between keratinocytes and the surrounding healthy connective tissue matrix. The results suggest that regardless of the surrounding matrix, LM-5 deposition appears to be critical for cell migration and BM zone organization during reepithelialization. Among the cytokines tested in vitro, TGFß and INF appear to act as potential upregulators of LM-5 expression.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Samples
Suction blisters were induced on the abdominal skin of 12 volunteers, aged 2266 years (
Experimental human mucosal wounds (full-thickness) were made in human palate as described previously (
A spontaneously transformed nontumorigenic human adult skin epithelial cell line that has maintained epithelial differentiation properties (HaCat) (
Immunohistochemical Studies
Immunostaining by an avidinbiotin technique (Vector Elite kit; Abbott, Chicago, IL) was performed as described previously (2-chain (
Immunofluorescence staining of integrins and BM zone components was performed as described earlier by 3ß1 integrin (MAb J143) (
6ß4 integrin (recognizes ß4 chain) (
2-chain of LM-5 (
Control stainings were performed without the primary antibodies and with nonimmune mouse/rabbit serum.
In Situ Hybridization
In situ hybridization was done on 10 4-day-old blister samples as described previously by 2-chain, a PstI-EcoRI fragment of clone L15 (bases 29953840) was subcloned into the pSP64 and pSP65 vectors in the sense and anti-sense orientations (
Immunoprecipitation Analyses
Normal human mucosal keratinocytes were isolated from surgical gingival biopsies. Keratinocytes (passage <6) were cultured as described ( (20 ng/ml; Boehringer Mannheim, Mannheim, Germany), INF
(1000 U/ml; Boehringer), and IL-1ß (5 U/ml; Boehringer) in basal keratinocyte growth medium for 24 hr, followed by a medium change with fresh cytokines and radiolabeling with 100 µCi/ml [35S]-methionine (trans 35S label, specific activity >1000 Ci/mmol; ICN Radiochemicals, Irvine, CA) for 24 hr. Integrins were immunoprecipitated as described in detail elsewhere (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Upregulation of the LM-5 2-Chain in Blister Wounds
In fresh suction blisters, the LM-5 2-chain was mostly visible in the blister floor, but some staining could also be seen in the basal keratinocytes of the detached epidermis (not shown). In 2-day-old suction blisters, the LM-5
2-chain was visible in the blister floor in areas not covered by regenerated epidermis, but also cytoplasmically in the keratinocytes at the blister roof, perhaps reflecting active synthesis of the LM-5
2-chain (Figure 1A). In 4-day-old blisters, staining of the LM-5
-chain was more intense in the areas covered by new epidermis than in normal skin (Figure 1BD) (Table 1.). At the leading edge of the regenerating epidermis, covering a distance of about 2050 cells from the tip, the LM-5
2-chain was expressed in the keratinocyte cytoplasm (Figure 1C and Figure 1D). Behind these cells, the LM-5
2-chain was present as a thin continuous line at the basement membrane zone (Figure 1C and Figure 1D). Table 1 shows a summary of the immunostaining results from 10 subjects. In 9-day-old blisters, the entire blister base was covered by a new acanthotic epidermis, and LM-5
2-chain staining was comparable to that of the controls (Figure 1E and Figure 1F).
|
|
The expression of the LM-5 2-chain in human blister wounds was then studied in 10 cases of 4-day-old blisters by in situ hybridization. In normal skin, no or only a very weak signal could be observed in basal keratinocytes (not shown). In regenerating epidermis, the signal for LM-5
2-chain mRNA was especially strong close to the leading edge, with a clear decreasing gradient towards the normal skin areas (Figure 1G and Figure 1H). A strong signal for LM-5
22-chain mRNA could also be seen close to the tip of the leading edge in the keratinocytes that were not in apparent contact with the underlying dermis. The activation of the LAMC2 gene during wound healing was independent of the age of the subjects (Table 1). The pattern of LM-5
2-chain mRNA expression was remarkably similar in the healing blisters of the subjects aged 2266 years.
LM-5 Receptors in Blister Wounds
In normal epidermis, 3ß1 integrin was localized around basal keratinocytes, especially in the cellcell contact areas (Figure 2A). Virtually no expression was seen in suprabasal cell layers. In 4-day-old blister wounds,
3ß1 integrin was expressed throughout the cell layers of the leading edge. Remarkably strong staining was seen in the cell extension of the leading keratinocytes (Figure 2B).
6ß4 Integrin was localized at the basal aspect of the basal keratinocytes of normal epidermis (Figure 2C). At the leading edge of healing 4-day-old blisters,
6ß4 was localized around basal and suprabasal keratinocytes (Figure 2D).
|
LM-5 in Full-thickness Mucosal Wounds
Using mucosal full-thickness wounds, we examined the distribution of the LM-5 2-chain under conditions in which keratinocytes are known to migrate through the clot (
2-chain was continuously present underneath the migrating keratinocytes of 3-day-old wounds, whereas LM-1 and collagen Types IV and VII were almost completely absent (Figure 3). The expression of immunoreactive LM-5 protein was higher in the wound area compared to the non-wounded site in oral mucosa (Figure 3).
|
Development of a BM Zone in a Cyst Cavity in Athymic Mice
Using the inclusion cyst model, we explored which component of the BM zone plays an initiating role when BM is deposited in inflammation-free connective tissue. In immature 2-day-old cysts of HaCat keratinocytes, the LM-5 2-chain was found both in peripheral cells and inside the cyst cavity (Figure 2F). Type VII and IV collagens were found only in cells inside the cyst (Figure 2G and Figure 2H, respectively). The immunolocalization pattern of
6ß4 integrin was similar to that of the LM-5
2-chain in 2-day-old cysts (Figure 2E). After 7 days the cysts appeared to be mature, with cells sloughing centrally. At this stage, all the components of the BM zone were localized around the cyst periphery (Figure 2IL). At that time, there were practically no staining signals in keratinocytes located inside the epithelial cysts.
Regulation of LM-5 Expression in Cultured Mucosal Keratinocytes
Because LM-5 appears to be upregulated during epidermal and mucosal wound healing, we wanted to examine whether the cytokines present during the different various stages of wound healing could influence LM-5 expression. Among the cytokines tested, TGFß and INF were found to upregulate the synthesis of LM-5 in cultured mucosal keratinocytes (Figure 4). Both of these cytokines also stimulated the biosynthesis of fibronectin, whereas they had no noticeable effect on the expression of ß1 integrins (Figure 4). TNF
slightly stimulated the synthesis of fibronectin, whereas it did not have any visible effect on LM-5 synthesis (Figure 4).
|
Because the association of LM-5 protein with the cell culture fraction of keratinocytes, it is not possible to quantitate all the LM-5 deposited by keratinocytes, and these results therefore provide only semiquantitative information about the regulation of LM-5 by different growth factors.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Regeneration of surface epithelium during wound healing takes place in a controlled fashion through several mechanisms, such as migration, proliferation, and differentiation (
Suction blisters have proved to be a convenient model for studies of wound healing under conditions in which the BM remains relatively intact, containing Type IV collagen, for example (
During reepithelialization, abundant deposition of LM-5 molecules was observed by immunohistochemistry under the regenerating epidermis of suction blisters. The in situ hybridization findings confirmed the new transcription of LM-5 molecules, showing that the most actively synthesizing cells were at the leading edge of the regenerating epidermis. Because the total length of the reepithelialization was about 100120 cells and the number of LM-5 2-chain mRNA-containing cells was 1530 on Day 4, the activation of the LAMC2 gene is relatively transient. The expression pattern of the LM-5
2-chain mRNA was similar in blisters in which remnants of LM-5 could be seen in the blister floor and in those in which LM-5 was absent. This indicates that activation of the LAMC2 gene is required even when LM-5 molecules already exist in the wound bed. It therefore appears that LM-5 molecules, in the form of broken anchoring filaments, are not used by the migratory keratinocytes, but instead they deposit new LM-5 molecules for interaction with the surrounding matrix. In full-thickness incisional mucosal wounds, the deposition of LM-5 occurs clearly before the deposition of the other BM zone components, such as Types IV and VII collagen or laminin-1, as shown here and in our earlier publication (
The 3ß1 and
6ß4 integrins, receptors for LM-5, localize in similar areas as LM-5, suggesting that keratinocytes might use either of these integrins as their LM-5 receptor (
6ß4 integrin could have two stages of interaction with LM-5 (
6ß4 integrin develop.
In the epidermal inclusion cyst model, LM-5 and its receptor, 6ß4 integrin, were seen to organize as early as Day 2, first at the cyst periphery, whereas there was no accumulation of Types IV or VII collagen. Previously, neither laminin-1 nor bullous pemphigoid antigen have been reported to be detectable in immature 2-day-old cysts but were reported to be present in mature 7-day-old cysts (
We found that TGFß and INF were able to induce the expression of the LM-5 protein in cultured mucosal keratinocytes, whereas there was no effect with IL-1ß or TNF
. This result is in accordance with the report by
. TGFß also induced the production of fibronectin, as reported previously (
appear to be the best regulators of LM-5 expression.
In conclusion, induction of the LM-5 gene and protein expression appears to be critical during reepithelialization, regardless of the injury or the microenvironment of the wounds. The LM-5 gene can be activated by TGFß and INF present in the wound fluids. LM-5 appears to be used as an intermediate bridging molecule between the
3ß1 or
6ß4 integrins of migrating keratinocytes and the wound matrix. At the later stages of healing, LM-5 could serve as the first anchoring element inhibiting cell migration and promoting the formation of stable contacts with BM components via hemidesmosomes.
![]() |
Acknowledgments |
---|
Supported by grants from the Medical Research Council of the Academy of Finland, the Medical Research Council of Canada, the Cancer Societies of Finland, Finland's Cancer Institute, and the Sigrid Juselius Foundation.
We thank Ms Annikki Huhtela for expert technical assistance, Dr Juha Tuukkanen for assisting in the image analysis of in situ hybridizations, and Dr Vesa Koivukangas for providing biopsy material.
Received for publication June 18, 1997; accepted October 13, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AutioHarmainen H, Sandberg M, Pihlajaniemi T, Vuorio E (1991) Basement membrane synthesis in developing placenta. Lab Invest 4:483-491
Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106:761-771[Abstract]
Carter WG, Ryan MC, Gahr PJ (1991) Epiligrin, a new cell adhesion ligand for integrin 3ß1 in epithelial basement membranes. Cell 65:599-610[Medline]
Champliaud MF, Lundstrom GP, Rousselle P, Nishiyama T, Keene DR, Burgeson RE (1996) Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment. J Cell Biol 132:1189-1198[Abstract]
Clark RAF (1995) Wound repair. Overview and general considerations. In Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. New York, Plenum Press, 3-50
GagnouxPalacios L, Vailly J, DurandClement M, Wagner E, Ortonne J-P, Menequzzi G (1996) Functional re-expression of laminin-5 in laminin-gamma2-deficient human keratinocytes modifies cell morphology, motility and adhesion. J Biol Chem 271:18437-18444
Grimwood RE, Baskin JB, Nielsen LD, Feris CF, Clark RAF (1988) Fibronectin extracellular matrix assembly by human epidermal cells implanted into athymic mice. J Invest Dermatol 90:434-440[Abstract]
Hertle MD, Jones PH, Groves RW, Hudson DL, Watt FM (1995) Integrin expression by human epidermal keratinocytes can be modulated by interferon-gamma, transforming growth factor-beta, tumor necrosis factor-alpha, and culture on a dermal equivalent. J Invest Dermatol 104:260-265[Abstract]
Hormia M, FalkMarzillier J, Plopper G, Tamura RN, Jones JC, Quaranta V (1995) Rapid spreading and mature hemidesmosome formation in HaCat keratinocytes induced by incubation with soluble laminin-5. J Invest Dermatol 105:557-561[Abstract]
Kainulainen T, AutioHarmainen H, Oikarinen A, Salo S, Tryggvason K, Salo T (1997) Abnormal distribution and synthesis of laminin-5 (kalinin) in oral lichen planus, epithelial dysplasias and squamous cell carcinomas. Br J Dermatol. 136:331-336[Medline]
Kallunki P, Sainio K, Eddy R, Byers M, Kallunki T, Sariola H, Beck K, Hirvonen H, Shows TB, Tryggvason K (1992) A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment. J Cell Biol 119:679-693[Abstract]
Kantor RRS, Mattes MJ, Loyd KO, Old LJ, Albino P (1987) Biochemical analysis of two cell surface glycoprotein complexes, very common antigen 1 and very common antigen 2. J Biol Chem 262:15158-15165
Kennel SJ, Foote LJ, Falcioni R, Sonnenberg A, Stringer CD, Crouse C, Hemler ME (1989) Analysis of the tumor-associated antigen TSP-180. J Biol Chem 264:15515-15521
Kiistala U (1968) Suction blister device for separation of viable epidermis from dermis. J Invest Dermatol 50:129-137[Medline]
Korang K, Christiano AM, Uitto J, Mauviel A (1995) Differential cytokine modulation of the genes LAMA3, LAMB3, and LAMC, encoding the constitutive polypeptides, alpha3, beta 3, gamma 2, of human laminin 5 in epidermal keratinocytes. FEBS Lett 368:556-568[Medline]
Kurpakus MA, Quaranat V, Jones JCR (1991) Surface relocation of alpha6beta4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing. J Cell Biol 115:1737-1750[Abstract]
Larjava H, Haapasalmi K, Salo T, Wiebe C, Uitto VJ (1996) Keratinocyte integrins in wound healing and chronic inflammation of the human periodontium. Oral Dis 2:77-86[Medline]
Larjava H, Peltonen J, Akiyama SK, Yamada SS, Gralnick HR, Uitto J, Yamada KM (1990) Novel function for beta-1 integrins in keratinocyte cell-cell interactions. J Cell Biol 110:804-815
Larjava H, Salo T, Haapasalmi K, Kramer RH, Heino J (1993) Expression of integrins and basement membrane components by wound keratinocytes. J Clin Invest 92:1425-1435[Medline]
Lavker RM, Sun T-T (1983) Rapid modulation of keratinocyte differentiation by external environment. J Invest Dermatol 80:228-237[Abstract]
Matsui C, Wang CK, Nelson CF, Bauer EA, Hoeffner WK (1995) The assembly of laminin-5 subunits. J Biol Chem 270:23496-23503
Niessen CM, Hogervorst F, Jaspars LH, de Melker AA, Delwel GO, Hulsman EH, Kuikman I, Sonnenberg A (1994) The alpha 6 beta 4 integrin is a receptor for both laminin and kalinin. Exp Cell Res 211:360-367[Medline]
Oikarinen A, Savolainen E-R, Tryggvason K, Foidart JM, Kiistala U (1982) Basement membrane components and galactosylhydroxylysyl glucosyl transferase in suction blisters of human skin. Br J Dermatol 106:257-266[Medline]
Pyke C, Salo S, Ralfkioer E, Romer J, Dano K, Tryggvason K (1995) Laminin-5 is a marker of invading cancer cells in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas. Cancer Res 55:4132-4139[Abstract]
Roberts CJ, Birkenmeier TM, McQuillan JJ, Akiyama SK, Yamada SS, Chen WT, Yamada KM, McDonald JA (1988) Transformation growth factor beta stimulates the expression of fibronectin and both subunits of the human fibronectin receptor by cultured human lung fibroblasts. J Biol Chem 263:4568-4592
SaarialhoKere U, Vaalamo M, Airola K, Niemi K-M, Oikarinen AI, Parks WC (1995) Interstitial collagenase is expressed by keratinocytes, that are actively involved in re-epithelization in blistering diseases. J Invest Dermatol 104:982-988[Abstract]
Salo T, Lyons G, Rahemtulla F, BirkedalHansen H, Larjava H (1991) Transforming growth factor ß1 up-regulates type IV collagenase expression in cultured human keratinocytes. J Biol Chem 266:11436-11441
Uitto J, Pulkkinen L, Christiano AM (1994) Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes. Review. J Invest Dermatol 103:39-46
Verrando P, Hsi BL, Yeh CJ, Pisani A, Serieyes N, Ortonne JP (1987) Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes. Exp Cell Res 170:116-128[Medline]
Verrando P, Lissitzky J-C, Sarret Y, Winberg JO, GeddeDahl T, Schmitt D, BrucknerTuderman L (1994) Keratinocytes from junctional epidermolysis bullosa do adhere and migrate on the basement membrane protein nicein through 3ß1 integrin. Lab Invest 71:567-574[Medline]