Characterization of the gene for human EMMPRIN, a tumor cell surface inducer of matrix metalloproteinases.

EMMPRIN (extracellular matrix metalloproteinase inducer) also known as CD147 and basigin, is a member of the immunoglobulin family that is present on the surface of tumor cells and stimulates nearby fibroblasts to synthesize matrix metalloproteinases. Using our EMMPRIN cDNA, we have isolated a cosmid clone that contains the human EMMPRIN gene. S1 analysis with a fragment of the gene clone and primer extension of the mRNA was performed to determine the transcription start site. PCR and sequence analysis have defined the exon/intron organization of the gene and show that it is highly conserved with the mouse EMMPRIN/basigin gene. About 950 bases of the 5'-flanking region were examined for transcription factor consensus binding sites, locating three SP1 sites and two AP2 sites. The transcription start site was found to be located in a CpG island. Elements in the proximal promoter region were conserved in the human and mouse genes.

A comparison of wire brush and diamond fraise superficial dermabrasion for photoaged skin. A clinical, immunohistologic, and biochemical study.

BACKGROUND: Superficial dermabrasion has a proven beneficial effect on photoaged skin, but little is known about the differences between the two major modalities used in dermabrasion, the diamond fraise (DF) and the wire brush (WB). OBJECTIVE: We compared the clinical, immunohistologic, and biochemical changes after superficial dermabrasion with DF and WB. METHODS: Eight photoaged patients (mean age, 68 years; range, 49 to 80 years) underwent facial dermabrasion to the level of the papillary dermis. Clinical assessments were performed at baseline and at 3 and 12 weeks after dermabrasion. Biopsy specimens were taken from both dermabraded halves at the same time points and assessed by routine histologic and immunohistologic examinations, western blot analysis, and radioimmunoassay. Scoring of intracellular and extracellular transforming growth factor-beta 1 was based on a semiquantitative ordinal scale (0 = no staining to 4 = maximum staining) in half-unit increments. The score for each specimen represents the average of values obtained from four high-power fields. RESULTS: Both methods of dermabrasion resulted in significant resolution of actinic keratoses, lentigines, and wrinkling. No statistical significance was noted between the two methods in regard to clinical efficacy. Significantly fewer milia occurred after DF than after WB. Solar elastosis decreased with both the WB and DF. Immunohistologic examination demonstrated a highly significant increase in papillary dermal fibroblast staining for amino terminal procollagen I (type I pN-collagen) at 3 weeks for both DF and WB compared to baseline. Staining at 12 weeks had decreased from the peak noted at week 3, but was still significantly increased from baseline. Western blotting of type I pN-collagen demonstrated a 5.4-fold (p = 0.01) increase from baseline at 3 weeks and a 4.9-fold (p = 0.002) increase at 12 weeks after dermabrasion with the WB. Similarly, the DF produced a 4.9-fold (p = 0.006) increase at 3 weeks and a 5.1-fold (p = 0.008) increase at 12 weeks after dermabrasion. Western blotting of amino terminal procollagen III (type III pN-collagen) showed a 6.1-fold (p = 0.07) increase from baseline at 3 weeks and a 3.9-fold (p = 0.04) increase at 12 weeks after dermabrasion with the DF. The WB showed a 3.8-fold (p = 0.07) increase from baseline at 3 weeks and a 5.1-fold (p = 0.05) increase at 12 weeks. Transforming growth factor-beta 1 demonstrated a significant increase in extracellular staining with DF (3.3 +/- 0.2) and WB (3.7 +/- 0.2) from baseline (1.2 +/- 0.2, p < 0.001) at 3 weeks. CONCLUSION: Superficial dermabrasion with DF and WP appears to be similarly efficacious in the treatment of photoaged skin. Significant increases in type I pN-collagen, type III pN-collagen, and TGF-beta 1 occurred in the papillary dermis after both types of dermabrasion. These results suggest that increased fibroblast activity and consequent collagen I and III synthesis underlie the clinical improvement.

Pilot histologic and ultrastructural study of the effects of medium-depth chemical facial peels on dermal collagen in patients with actinically damaged skin.

BACKGROUND: Chemical peels are employed for a variety of benign and premalignant skin disorders. OBJECTIVE: We compared clinical and histologic features with ultrastructural changes that occur after medium-depth chemical facial peel. METHODS: Three men with actinically damaged facial skin underwent a single 35% trichloroacetic acid peel. Biopsy specimens were taken before the peel, and 2 weeks and 3 months after the peel, for histologic examination, electron microscopy, and gel electrophoresis to assess total collagen type I content. RESULTS: Clinical resolution of actinic damage corresponded with restoration of epidermal polarity. Collagen type I was markedly increased after the peel. Characteristic ultrastructural features of skin after peeling include markedly decreased epidermal intracytoplasmic vacuoles, decreased elastic fibers, and increased activated fibroblasts. CONCLUSION: Electron microscopic studies after a medium-depth chemical peel of photodamaged skin reveal more profound changes than those seen histologically.

Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3.

To elucidate structure-function relationships of stromelysin-3, a putative matrix metalloproteinase originally identified at the tumor-stromal cell interface in breast carcinomas, the human cDNA was expressed in mammalian cells, and its products were isolated and characterized. In stably transfected cells, stromelysin-3 was recovered as a complex mixture of species ranging in size from approximately 20 to 65 kDa. Among these products, a major 45-kDa species with an N terminus of Phe98 and an intact C-terminal domain was identified as a true endopeptidase on the basis of its ability to cleave the bait region of alpha 2-macroglobulin between Phe684 and Tyr685, a site identical to that recognized by stromelysin-1. However, unlike stromelysin-1 or other members of the matrix metalloproteinase family, the mature form of stromelysin-3 was unable to hydrolyze a range of extracellular matrix molecules associated with either the basement membrane or interstitium. To probe for alternate substrates among tumor cell-derived products, purified stromelysin-3 was incubated with [35S]methionine-labeled medium conditioned by the breast cancer cell line, MCF-7. Under these conditions, a single, tumor cell-derived protein was hydrolyzed as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Following anion-exchange chromatography and preparative gel electrophoresis, the stromelysin-3 substrate was identified by N-terminal sequencing as the serine proteinase inhibitor, alpha 1-proteinase inhibitor. Further studies demonstrated that stromelysin-3 rapidly destroyed the antiproteolytic function of alpha 1-proteinase inhibitor by cleaving the antiproteinase at a distinct site between Ala350 and Met351 within the reactive-site loop. Together, these data not only demonstrate that human stromelysin-3 acts as a powerful endopeptidase with a restricted substrate specificity distinct from all other matrix metalloproteinases, but also serve to identify serine proteinase inhibitors as potential physiologic targets at sites of extracellular matrix remodeling.

Clinical improvement following dermabrasion of photoaged skin correlates with synthesis of collagen I.

BACKGROUND AND DESIGN: The ability of superficial dermabrasion to improve clinical features of photoaged skin is well known, but the specific biological mechanisms involved are poorly understood. The so-called repair zone, as visualized by routine histologic examination, has been attributed to new collagen formation within the papillary dermis and may be responsible for clinical improvement following dermabrasion. We investigated molecular and histologic events occurring in dermabraded skin and correlated them with clinical improvement. Ten photoaged patients (mean age, 59 years) underwent facial dermabrasion to the level of the papillary dermis. Clinical severity of photoaging was graded in a blinded manner at baseline and 12 weeks after dermabrasion. Biopsy specimens obtained at baseline and 3 and 12 weeks after dermabrasion were analyzed histologically and by in situ hybridization for fibroblast procollagen I mRNA, immunohistologically and by Western blotting with a monoclonal antibody specific for the aminoterminal cleavage site of procollagen I. RESULTS: Masson's trichrome staining demonstrated an increase in collagen from baseline (as an upper dermal band in the dermabrasion "repair zone") at 3 and 12 weeks' postdermabrasion. Immunohistologic examination demonstrated papillary dermal fibroblast staining for procollagen I at baseline that increased by threefold at 3 weeks' postdermabrasion and by 1.5-fold at 12 weeks' postdermabrasion. Western blotting demonstrated an average-fold increase in pN collagen I of 4.2 +/- 1.5 at 3 weeks and of 2.7 +/- 0.7 at 12 weeks. By in situ hybridization, baseline levels of procollagen I mRNA in papillary dermal fibroblasts increased sixfold at weeks 3 and 12 postdermabrasion. Increase in procollagen I mRNA correlated with clinical improvement, ie, reduction in wrinkling. CONCLUSION: Superficial dermabrasion clinically improves photoaged skin, and this improvement correlates strongly with increased collagen I gene expression.

Increased expression of matrix metalloproteinase-3 (stromelysin-1) in cultured fibroblasts and basal cell carcinomas of nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS), an autosomal dominant disorder, is characterized by the development of numerous cutaneous basal cell carcinomas (BCCs). To better characterize this disorder, we examined the expression of matrix metalloproteinase-3 (MMP-3; also known as stromelysin-1) in BCC tumor specimens, adjacent normal skin, and fibroblasts isolated from the normal-appearing skin of NBCCS patients. Three of three BCC tumors obtained from NBCCS patients overexpressed MMP-3 mRNA. In contrast, only 25% of BCC specimens in patients without NBCCS demonstrated overexpression of MMP-3 mRNA. Moreover, fibroblasts isolated and cultured from all nine uninvolved skin specimens of NBCCS patients overexpressed MMP-3 mRNA. MMP-3 mRNA was not detected or was detected at very low levels in normal skin and fibroblast cultures isolated from normal skin in nonsyndrome patients.

Immediate X-ray-inducible responses from mammalian cells.

It has been nearly 6 years since we reported the induction of new proteins in human normal and tumor cells after ionizing radiation. Since that time there has been an explosion of new data and ideas from a number of laboratories regarding the immediate responses of human cells to ionizing radiation. The data are, however, extremely difficult to interpret since researchers are using confluence-arrested, log-phase, normal or tumor cells, and are exposing these to a variety of doses of ionizing radiation. It is especially difficult to interpret data from cells that are exposed to supralethal doses of ionizing radiation. Thus this session of the workshop entitled "Molecular, Genetic, and Cellular Basis of Radioresistance at Low Doses: A Case Of Inducible Repair?" concentrated on inducible responses (both late and immediate) of human cells exposed to physiological doses of ionizing radiation. A major focus of future research in this field must be directed toward the function(s) of these inducible proteins and the expression of genes in DNA repair, cell cycle progression (especially radiation-induced cell progression delays) and/or cell death, including apoptosis.

Increased expression of stromelysin-3 in basal cell carcinomas.

We examined the expression of two groups of matrix metalloproteinases (MMPs), stromelysin and interstitial collagenase, in human skin cancer by northern blot analysis and in situ hybridization. Stromelysin-3 (ST-3) mRNA was overexpressed more than tenfold in 17 of 19 (89%) specimens of basal cell carcinoma (BCC) but in only three of 13 (23%) cutaneous squamous cell carcinomas (SCCs). Stromelysin-1 and -2 (ST-1/2) mRNA was overexpressed in three of 19 (16%) BCC and three of 13 (23%) SCC. Collagenase mRNA was overexpressed in nine of 19 (47%) BCC and three of 13 (23%) SCC. No mRNA for ST-3, ST-1/2, or collagenase was detected by northern analysis in 21 specimens of adjacent normal skin. Because of these findings, we examined the specific location of the ST-3 mRNA in BCC specimens by in situ hybridization. ST-3 mRNA was particularly abundant in the characteristic stroma adjacent to the invasive basaloid tumor islands of the BCC and absent in the malignant cells. Moreover, ST-3 mRNA was expressed and induced by phorbol ester treatment in adult dermal fibroblasts but not in keratinocytes. In vitro studies have shown that MMPs are involved in the degradation of extracellular matrix molecules. Our finding of ST-3 mRNA overexpression in 17 of 19 (89%) BCC specimens is consistent with a role for this molecule in local invasion of stroma by BCC. Our in situ hybridization data suggested that while ST-3 is not expressed by malignant basal cells themselves, these tumor cells may induce the expression of ST-3 in adjacent nonmalignant stromal elements such as fibroblasts.

Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid)

BACKGROUND: Topical tretinoin (retinoic acid) modifies fine wrinkles and certain other features of human skin damaged by exposure to the sun (photodamage), but histologic changes do not account for this improvement. In mice with photodamage induced by ultraviolet light, effacement of fine wrinkles by tretinoin is correlated with dermal collagen synthesis but not with histologic changes. We investigated whether collagen synthesis was reduced in photodamaged human skin and, if so, whether it could be restored by treatment with topical tretinoin. METHODS: Biopsies of photodamaged skin from the extensor aspect of the forearm and skin from the buttocks, which had been protected from the sun, were performed on 26 healthy subjects. In addition, 29 patients with photodamaged skin were treated for 10 to 12 months with a daily application of 0.1 percent tretinoin cream (15 patients) or vehicle cream (14 patients). Skin-biopsy specimens obtained at base line and after treatment were assessed immunohistologically for evidence of dermal collagen I formation (collagen synthesis). RESULTS: Collagen I formation was 56 percent less in the papillary dermis of photodamaged skin than in skin protected from the sun (P < 0.001) and was correlated with the clinical severity of photodamage (r = -0.58, P = 0.002). Treatment of photodamaged skin with tretinoin produced an 80 percent increase in collagen I formation, as compared with a 14 percent decrease in collagen formation with the use of vehicle alone (P = 0.006). CONCLUSIONS: The formation of collagen I is significantly decreased in photodamaged human skin, and this process is partly restored by treatment with tretinoin.

Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin.

We report a novel method to culture chick embryo osteoblasts in vitro. Primary cells were grown from explants of calvaria and then cultured within alginate polymer beads. Enriched cultures of primary osteoblasts were obtained because these cells grow readily within alginate beads but other cell types present in the initial outgrowth from calvarial fragments, such as fibroblasts, do not. A reproducible bone cell phenotype was observed in calvarial cells cultured in the alginate polymer for as long as 8 months. Alginate is a uronic acid monomer that reversibly polymerizes based on the presence or absence of divalent cations. Osteoblasts derived from the alginate beads elaborated and mineralized an extracellular matrix in vitro that contained fibronectin, type III collagen, and type I collagen. The synthesis and deposition of these matrix molecules was also demonstrated in the chick embryo calvaria in vivo. Together, these in vitro and in vivo observations provide the first evidence that type III collagen and fibronectin colocalize with type I collagen during the development of avian membranous bone. They also indicate that the phenotype of chick embryo osteoblasts can be expanded to include the synthesis of fibronectin and type III collagen.

Different mechanisms decrease hepatic collagen and albumin production in fasted rats.

Weight loss is correlated with a specific decrease in collagen synthesis in extrahepatic tissues, mainly through modulation of mRNA levels. Here, we investigated the response to weight loss in the rat liver. Male rats were either fed ad libitum or fasted for 92 hr; fasted animals lost approximately 20% of their initial body weight. Following i.p. injection of [5-3H]proline, hepatic collagen was extracted and de novo collagen production was measured. There was a decrease in the specific radioactivities of purified hepatic collagen (-75%) and albumin (-70%) relative to total hepatic protein, indicating that production of both of these proteins was specifically decreased. In fasted animals, the absolute hepatic collagen production was markedly decreased (-60%), while changes in absolute hepatic protein production were small (-15%). Using hybridization with specific DNA probes, we found that fasting causes about a 70% decrease in albumin mRNA, but the quantities of hepatic procollagen alpha 1(I) and alpha 2(I) mRNAs were unchanged. These results are consistent with regulation of albumin production during fasting by modulation of mRNA levels. The inhibition of hepatic collagen production in fasted animals, however, appears to be modulated at a posttranscriptional level or may result from increased degradation. This response differs from the pretranslational regulation of collagen synthesis in extrahepatic tissues during fasting. Furthermore, our results suggest that decreased body weight could be a potentially complicating variable in studies of collagen metabolism and fibrogenesis in the liver.

Uncoupled expression of mRNAs for alpha 1(I) and alpha 2(I) procollagen chains in chemically transformed Syrian hamster fibroblasts.

Syrian hamster embryo fibroblasts transformed by 4-nitroquinoline-1-oxide (NQT-SHE cells) failed to synthesize the pro-alpha 1(I) subunit of type I procollagen but continued to synthesize altered forms of the other subunit, pro-alpha 2(I) (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). This was unusual, since synthesis of the two subunits generally is coordinately regulated. Present experiments using cell-free translation and hybridization of RNA from normal and transformed Syrian hamster fibroblasts with labeled pro-alpha 1(I) DNA probes show that mRNA for pro-alpha 1(I) is absent from the transformant. In contrast, dot-blot and Southern blot hybridizations of cellular DNAs with pro-alpha 1(I) DNA probes demonstrated that the transformed cells contained pro-alpha 1(I) gene sequences and that the gross structure of the gene was unchanged by transformation. mRNA for the other type I procollagen subunit, pro-alpha 2(I), was present in transformed cells and the major collagenous polypeptide translated from this RNA migrated like the normal pro-alpha 2 subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translated procollagen chain was cleaved to an alpha 2(I)-sized collagen chain by pepsin at 4 degrees C. These studies provide a molecular basis for the observed collagen phenotype of NQT-SHE cells.

Cyclic AMP-independent processes mediate Kirsten sarcoma virus-induced changes in collagen production and other properties of cultured cells.

Previous studies suggested that the decreased collagen production observed in Kirsten sarcoma virus (Ki-MSV)-transformed BALB 3T3 cells could be reversed by treating cells with Bt2cAMP. We examined the relationship between intracellular cAMP, collagen production, and other properties in NRK and BALB 3T3 cells transformed by Ki-MSV. Two 3T3 transformants (Ki-3T3-234 and Ki-3T3Cl1) had lower cAMP levels than nontransformed cells. The level in a temperature-sensitive transformant, tsKi-3T3-714, was similar to the level in its parent, 3T3-714, and when it was shifted to a temperature nonpermissive for transformation (40 degrees C), intracellular cAMP did not increase although the growth and morphological properties were normal. The relative rate of collagen production also increased to the normal level. These results indicate that transformation-induced changes were regulated independently of cAMP. Further observations supported this conclusion. Intracellular cAMP in a flat revertant of Ki-3T3Cl1 was lower, rather than higher, than in the transformant, although the relative rate of collagen production was higher. Treatment of Ki-3T3-234 and tsKi-3T3-714 with cholera toxin plus isobutylmethylxanthine increased intracellular cAMP concentrations to 2-20 times the level in untreated cells, levels much higher than in nontransformed 3T3. In spite of this, collagen production was not increased by these agents in tsKi-3T3-714 and it was only partially restored in Ki-3T3-234 relative to the level in the nontransformed cells. In contrast, these agents inhibited growth on a substratum or in soft agar and produced a flattened morphology in both lines. Similarly, collagen production in transformed NRK cells (K-NRK) was only 3% of normal but treatment with Bt2cAMP or cholera toxin plus isobutylmethylxanthine increased production to only one-third the normal level while increasing cAMP to four times the normal level. We conclude that in Ki-MSV-transformed BALB 3T3 cells, changes in cAMP may be secondary effects and not related to maintenance of the transformed phenotype. The high levels of cAMP induced by exogenous agents may act on similar targets as those affected by transformation, but reversal of the transformed phenotype by these agents probably occurs by a different mechanism than that originally used to impose the changes.

Regulation of Neurospora crassa nucleosidases by some environmental factors.

Enzymic cleavage of N-glycosidic bonds of AMP, GMP, and inosine by the cell-free extracts of Neurospora crassa has been studied. The enzymic activities with these substrates appear to be discrete from one another. The levels of these enzymes in the cell vary with age, and are dependent upon the inoculum size, aeration rate, and phosphate level in the medium. Glucose (or ribose) controls the phosphate-mediated repression of all the three nucleosidases of this fungus.