Identification of different isoforms of 14-3-3 protein family in human dermal and epidermal layers

We have previously demonstrated a high level of stratifin, also known as 14-3-3 sigma in differentiated keratinocyte cell lysate and conditioned medium (CM). In this study, we asked the question of whether other 14-3-3 isoforms are expressed in human dermal fibroblasts, keratinocytes, intact dermal and epidermal layers of skin. In order to address this question, total proteins extracted from cultured cells or skin layers were subjected to western blot analysis using seven different primary antibodies specific to well-known mammalian isoforms, beta, gamma, epsilon, eta, sigma, tau, and zeta of 14-3-3 protein family. The autoradiograms corresponding to each isoform were then quantified and compared. The results revealed the presence of very high levels of all seven isoforms in cultured keratinocyte and conditioned medium. With the exception of tau isoform, other 14-3-3 isoforms were also present in intact epidermal layer of normal skin. The profile of 14-3-3 proteins in whole skin was similar to that of epidermis. In contrast, only gamma 14-3-3 isoform, was present in dermal layer obtained from the same skin sample. On the other hand, cultured fibroblasts express a high level of beta, epsilon, gamma and eta and a low level of zeta and tau, but not sigma isoform. However, the levels of 14-3-3 epsilon, gamma and eta were barely detectable in fibroblast conditioned medium. Further, we also used immunohistochemical staining to identify the 14-3-3 isoform expressing cells in human skin sections. The finding revealed different expression profile for each of these isoforms mainly in differentiated keratinocytes located within the layer of lucidum. However, fibroblasts located within the dermal layer did not show any detectable levels of these proteins. In conclusion, all members of 14-3-3 proteins are expressed by cells of epidermal but not dermal layer of skins and that these proteins are mainly expressed by differentiated keratinocytes.     

Profile of exosomes related proteins released by differentiated and undifferentiated human keratinocytes

Our group has previously demonstrated the capacity of human keratinocytes to release 14‐3‐3σ into conditioned medium through the mechanism of exosome externalization. In this study the release of other proteins through the same mechanism and the differences in the profiles of 14‐3‐3 proteins between differentiated (diff‐K) and undifferentiated keratinocytes (undiff‐K) were investigated. The stimulatory effect of other 14‐3‐3 isoforms on the expression of MMP‐1 in dermal fibroblasts was also evaluated. Exosomes isolated from undiff‐K (low Ca²⁺) and diff‐K (high Ca²⁺) were subjected to proteomic and Western blot analysis. The results showed that more than 50 different cytoplasmic proteins including all seven 14‐3‐3 protein isoforms (β, σ, η, ε, τ, ζ, and γ) were released from diff‐K through the mechanism of exosome externalization. However, in exosomes of undiff‐K only four of the 14‐3‐3 protein isoforms (β, η, ζ, and γ) were detected. Ca²⁺ treatment increased the release of exosomes from undiff‐K by at least two times relative to the control. Consistent with this finding, the stimulatory effect of exosomes containing 14‐3‐3σ from diff‐K had higher MMP‐1 stimulatory effect in fibroblasts relative to those exosomes isolated from undiff‐K. MMP‐1 stimulatory effect of recombinant 14‐3‐3β and η, tested in this study, in dermal fibroblasts, suggests additional anti‐fibrogenic factors other than 14‐3‐3σ. In conclusion, keratinocytes release many proteins through the mechanism of exosome externalization from which some such as 14‐3‐3 isoforms may function as extracellular matrix (ECM) modulating factors for dermal fibroblasts. These findings revealed the presence of a novel mechanism by which keratinocytes can potentially interact with fibroblasts. J. Cell. Physiol. 221: 221–231, 2009. © 2009 Wiley‐Liss, Inc     

Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin

Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for clinical intervention in controlling excessive wound healing in fibrotic conditions.     

Inactivation of 14-3-3 σ by promoter methylation correlates with metastasis in nasopharyngeal carcinoma

14-3-3 σ, the downstream target of p53, is a negative regulator of cell cycle G2-M phase checkpoint in response to DNA damage. Our previous comparative proteomics study showed that 14-3-3 σ was downregulated or lost in nasopharyngeal carcinoma (NPC) tissue compared with non-cancerous nasopharyngeal epithelial tissue (NNET). In this study, we further investigated for the epigenetic mechanism of 14-3-3 σ inactivation. Methylation-specific PCR showed 14-3-3 σ promoter methylation in 100% of analyzed NPC cell lines (4/4) but not in immortalized human nasopharyngeal epithelial cell line NP69. Treatment of the four NPC cell lines with the methyltransferase inhibitor 5-aza-2′-dC resulted in the demethylation and upregulation of 14-3-3 σ. In tissues, 14-3-3 σ promoter methylation occurred at a higher frequency in NPC, 63/75 (84%), compared to adjacent NNET, 7/25 (28%), and fully methylated 14-3-3 σ promoter was detected in NPC but not in any of adjacent NNET. RT-PCR, Western blotting, and immunohistochemistry showed that 14-3-3 σ expression was downregulated or lost in NPC with methylation, and there was a negative correlation between the expression levels and methylation statuses of 14-3-3 σ gene. In addition, the patients with methylated 14-3-3 σ presented a higher frequency of lymph node and distant metastasis, and an advanced clinical stage, and overexpression of 14-3-3 σ in NPC cell line 5-8F with high metastatic potential was able to inhibit its in vitro invasive ability. Our data are the first to show that 14-3-3 σ is frequently inactivated by promoter methylation in NPC and this aberrant methylation correlates with lymph node and distant metastasis. J. Cell. Biochem. 106: 858–866, 2009. © 2009 Wiley-Liss, Inc.     

Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin

As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14-3-3 sigma (also known as stratifin), a keratinocyte-releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte-derived soluble factors. By using an in vitro keratinocyte-fibroblast co-culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte-conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose-dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte-derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal-mesenchymal communication.     

Establishment of 3D organotypic cultures using human neonatal epidermal cells

This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air-liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 mum) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes.     

The variable C-terminus of 14-3-3 proteins mediates isoform-specific interaction with sucrose-phosphate synthase in the yeast two-hybrid system

Sucrose-6-phosphate synthase (SPS) is a target for 14-3-3 protein binding in plants. Because several isoforms of the 14-3-3 protein are expressed in plants, I investigated which isoforms have the ability to bind SPS. Two 14-3-3 isoforms (T14-3d and a novel isoform designated T14-3 g) were found to interact with SPS from tobacco (Nicotiana tabacum L.) in a two-hybrid screen. To further address the question of isoform specificity of 14-3-3s, four additional isoforms were tested for their ability to interact with SPS in the yeast two-hybrid system. The results clearly revealed large differences in affinity between individual 14-3-3 isoforms toward SPS. Deletion analysis suggested that these differences were mediated by the variable C-terminus of 14-3-3s. Site-directed mutagenesis of candidate 14-3-3 binding sites on SPS demonstrated that interaction could be independent of a phosphorylated serine residue within conserved binding motifs in the yeast system. These findings suggest that the large number of 14-3-3 isoforms present in plants reflects functional specificity.     

Downregulation of 14-3-3 Proteins in a Kainic Acid-Induced Neurotoxicity Model

The 14-3-3 proteins are among the most abundant proteins expressed in the brain, comprising about 1% of the total amount of soluble brain proteins. Through phosphoserine- and phosphothreonine-binding motifs, 14-3-3 proteins regulate many signaling proteins and cellular processes including cell death. In the present study, we utilized a well-known kainic acid (KA)-induced excitotoxicity rat model and examined the expression of 14-3-3 and its isoforms in the frontal cortex of KA-treated and control animals. Among the different 14-3-3 isoforms, abundant levels of eta and tau were detected in the frontal cortex, followed by sigma, epsilon, and gamma, while the expression levels of alpha/beta and zeta/delta isoforms were low. Compared to the control animals, KA treatment induced a significant downregulation of the overall 14-3-3 protein level as well as the levels of the abundant isoforms eta, tau, epsilon, and gamma. We also investigated two 14-3-3-interacting proteins that are involved in the cell death process: Bcl-2-associated X (BAX) and extracellular signal-regulated kinase (ERK). Both BAX and phosphorylated ERK showed increased levels following KA treatment. Together, these findings demonstrate an abundance of several 14-3-3 isoforms in the frontal cortex and that KA treatment can cause a downregulation of 14-3-3 expression and an upregulation of 14-3-3-interacting proteins BAX and phospho-ERK. Thus, downregulation of 14-3-3 proteins could be one of the early molecular events associated with excitotoxicity. This could lead to subsequent upregulation of 14-3-3-binding proteins such as BAX and phospho-ERK that contribute to further downstream apoptosis processes, eventually leading to cell death. Maintaining sufficient levels of 14-3-3 expression and function may become a target of therapeutic intervention for excitotoxicity-induced neurodegeneration.     

Extracellular 14-3-3 from human lung epithelial cells enhances MMP-1 expression

Airway remodelling in asthma involves various mediators modulating the production/breakdown of collagen by lung fibroblasts. Matrix metalloproteinase-1 (MMP-1) plays an important role in collagen breakdown. We recently showed that epithelial cell-derived extracellular form of 14-3-3σ is an important inducer of MMP-1 expression in skin fibroblasts. Thus, we hypothesized that 14-3-3 proteins are important regulators of MMP-1 expression in the respiratory airway. We examined the presence of extracellular 14-3-3 proteins in conditioned media obtained from primary lung epithelial cells, A549 and HS24 cells, and their effect on MMP-1 expression by lung fibroblasts (IMR-90). In addition, we evaluated IMR-90 response to 14-3-3 proteins in the presence of transforming growth factor-β(1) (TGF-β(1)), a cytokine known to decrease MMP-1 expression by fibroblasts. Extracellular 14-3-3α/β, but not -σ, is released by the human-derived lung epithelial cell lines, A549 and HS24. Unlike dermal fibroblasts, IMR-90 cells do not produce MMP-1 in response to 14-3-3σ. Conversely, MMP-1 production was induced following treatment of IMR-90 with recombinant or lung epithelial cell-derived 14-3-3α/β. These findings were also confirmed using primary human bronchial epithelial cells and lung fibroblasts obtained from non-asthmatic patients. The MMP-1-inducing effect of 14-3-3α/β on IMR-90 was not inhibited by TGF-β(1). Lung epithelial cell-derived 14-3-3α/β has a potent MMP-1-inducing effect on airway fibroblasts. Modulation of MMP-1 by 14-3-3α/β, may be important in the alteration of collagenase production associated with airway remodelling in obstructive lung diseases. Our data indicate that 14-3-3 proteins may be potential targets for future therapeutic strategies aimed at modulating tissue remodelling in asthma.     

An obligatory heterodimer of 14-3-3beta and 14-3-3epsilon is required for aldosterone regulation of the epithelial sodium channel

Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323-16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3beta with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3beta alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3beta knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3epsilon, a second aldosterone-induced isoform. As observed previously for 14-3-3beta knockdown, small interfering RNA-induced reduction of 14-3-3epsilon markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3beta and 14-3-3epsilon were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3beta or 14-3-3epsilon reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, beta and epsilon, interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport.     

Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis.

14-3-3 proteins are a family of homologous eukaryotic molecules with seven distinct isoforms in mammalian cells. Isoforms of 14-3-3 proteins interact with diverse ligands and are involved in the regulation of mitogenesis, cell cycle progression, and apoptosis. However, whether different 14-3-3 isoforms are responsible for distinct functions remains elusive. Here we report that multiple isoforms of 14-3-3 proteins were capable of binding to several ligands, Bad, Raf-1, and Cbl. In a functional assay of 14-3-3 isoforms, all mammalian 14-3-3 isoforms could inhibit Bad-induced apoptosis. Thus, 14-3-3 function in regulating one of its ligands, Bad, is conserved among mammalian isoforms. We addressed whether 14-3-3 isoforms are differentially expressed in tissues, which may in part determine isoform-specific interactions. In situ hybridization revealed that 14-3-3zeta was present in most tissues tested, but sigma was preferentially expressed in epithelial cells. Thus, isoforms of 14-3-3 can interact and control the function of selected protein ligands, and differential tissue distribution of 14-3-3 isoforms may contribute to their specific interactions and subsequent downstream signaling events.     

Inhibitory effect of 14-3-3 proteins on serum-induced proliferation of cardiac fibroblasts

Proliferation in cardiac fibroblasts (CFs) can be induced by a wide variety of growth factors that recruit multiple signal transduction pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C. As a family of dimeric phophoserine-binding proteins, 14-3-3s are associated with a multitude of proteins that regulate signal transduction, apoptosis and checkpoint control pathways. However, it remains unknown whether the 14-3-3 proteins play an active role in cardiac proliferation and alter their expression patterns in response to growth factors in CFs. R18 peptide, an isoform-independent 14-3-3 inhibitor, was used to disrupt 14-3-3 function by adenovirus-mediated transfer of R18-EYFP (AdR18). Our results demonstrate that the 14-3-3 isoforms gamma, zeta and epsilon were highly expressed in CFs and the expression of 14-3-3 epsilon was elevated following serum stimulation. Inhibition of 14-3-3 proteins by AdR18 potentiated mitogen-induced DNA synthesis in CFs. This potentiation was presumably due to the increased inactivated glycogen synthase kinase-3 beta by Ser9 phosphorylation and nuclear factor of activated T-cell nuclear accumulation. However, AdR18 had no effect on extracellular signal-regulated kinase phosphorylation and reduced p70 S6 kinase (p70S6K) phosphorylation upon mitogenic stimulation. Furthermore, though R18 can block 14-3-3 binding abilities, it did not affect the serum-induced upregulation of 14-3-3 epsilon protein. Collectively, these findings reveal that the expression of 14-3-3 epsilon can be upregulated by serum in CFs and 14-3-3s may exert an inhibitory effect on serum-induced proliferation.     

Reduction of 14-3-3 proteins correlates with increased sensitivity to killing of human lung cancer cells by ionizing radiation

The 14-3-3 proteins have a wide range of ligands and are involved in a variety of biological pathways. Importantly, 14-3-3 proteins are known to be overexpressed in some human lung cancers, suggesting that they may play a role in tumorigenesis. Here we examined 14-3-3 expression in several lung cancer-derived cell lines and found that four of the seven 14-3-3 isoforms, beta, epsilon, theta and zeta, were highly expressed in both lung cancer cell lines and normal lung fibroblasts. Two isoforms, sigma and gamma, were present only at very low levels. Immunoprecipitation data showed 14-3-3zeta could bind to CDC25C in irradiated A549 cells, and suppression of 14-3-3zeta in A549 cells with antisense resulted in a decrease in CDC25C localization in cytoplasm and CDC2 phosphorylation on Tyr15. As a consequence, CDC2 activity remained elevated which resulted in release from radiation-induced G(2)/M-phase arrest. Moreover, 16% 14-3-3zeta antisense-transfected cells underwent apoptosis when exposed to 10 Gy ionizing radiation. These data indicate that 14-3-3zeta is involved in G(2) checkpoint activation and that inhibition of 14-3-3 may be a useful approach to sensitize human lung cancers to ionizing radiation.     

PRAS40与14-3-3蛋白各亚型相互作用的酵母双杂交系统检测

PRAS40是近几年新发现的Akt作用底物,14-3-3结合蛋白。为确定PRAS40与14-3-3蛋白7种亚基间相互作用关系,利用gateway方法构建用于酵母双杂交系统的诱饵质粒pEG-PRAS40及转录激活质粒pJG-PRAS40,将PRAS40和14-3-3各亚型质粒分别作为诱饵蛋白质粒及转录激活质粒共转化酵母细胞EGY48,通过氨基酸营养缺陷生长实验及β-半乳糖苷酶显色反应分析两种蛋白相互作用程度。酶切鉴定证实成功地构建了pEG-PRAS40和pJG-PRAS40质粒,酵母双杂交实验结果显示PRAS40可以和14-3-3亚型tau,beta,zeta及epsilon相结合,epsilon较强,beta和zeta次之,tau较弱。此结果将为深入研究PRAS40与14-3-3蛋白生物学功能及发现药物靶标奠定基础。     

The up-regulation of 14-3-3 proteins in Smad4 deficient epidermis and hair follicles at catagen

Each postnatal hair follicle (HF) perpetually goes through three phases: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still largely unknown. Our previous study shows that the keratinocyte specific Smad4 knockout mice exhibit progressive alopecia due to the mutant HFs failure to undergo programmed regression. To investigate the detailed molecular events controlling this process, the protein profiles of Smad4 mutant and control epidermal and HF keratinocytes were compared using 2-D difference gel electrophoresis (2-D DIGE) proteomic analysis. Eighty-six differentially expressed protein spots were identified by MALDI-TOF/TOF MS or ESI-MS/MS as 72 proteins, of which 29 proteins were found to be changed during the anagen-catagen transition of HFs in Smad4 mutants compared with the controls. The differentially expressed proteins represent a wide spectrum of functional classes such as keratin, the cytoskeleton, cellular growth and differentiation, ion combination and transfer, protein enzymes. Notably, we found that the 14-3-3sigma protein together with the 14-3-3zeta and 14-3-3beta proteins were significantly down-regulated only in wild-type keratinocytes but not in Smad4 mutant keratinocytes during the catagen phase, suggesting that increased expression of 14-3-3 proteins might contribute to the blockade of catagen initiation in Smad4 deficient HFs.     

Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase

The highly conserved family of 14-3-3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 suggest functional isoform specificity of 14-3-3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14-3-3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14-3-3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14-3-3 isoforms. We found that 14-3-3C is unavailable in dark harvested barley leaf extract and 14-3-3A is functionally not capable to efficiently inhibit NR activity, leaving 14-3-3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14-3-3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14-3-3A to the alternative residue, as found in 14-3-3B and 14-3-3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14-3-3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14-3-3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14-3-3 isoforms as well as binding affinity determine isoform-specific regulation of NR activity.     

Insulin-like growth factor-II/mannose 6 phosphate receptors facilitate the matrix effects of latent transforming growth factor-β1 released from genetically modified keratinocytes in a fibroblast/keratinocyte co-culture system

This study was conducted to explore the mechanism of activation of transforming growth factor-β1 (TGF-β1) which is critical to its role in many physiological and pathological conditions. To date, almost all reports concerning TGF-β1 activation delineated that release of mature TGF-β1 from latency associated protein (LAP) is required for its activation. We report that latent TGF-β1 (LTGF-β1) released from TGF-β1 genetically modified keratinocytes grown in the top chamber of a co-culture system functions as a fibrogenic factor through interaction with insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors of human dermal fibroblasts grown in the lower chamber of this system. Following successful transduction, the pLin-LTGF-β1 vector was amplified in PA317 packaging cells which possess viral structural proteins for vector in the presence of neomycin. Conditioned medium derived from packaging cells containing competent viral particles was then used to transduce either keratinocytes or fibroblasts grown in the upper chamber of a co-culture system, in which a 0.4 μm porous membrane separates the two chambers. In this way, LTGF-β1 produced by transduced cells in the upper chamber is released and diffuses into the lower chambers where dermal fibroblasts are grown. Conditioned medium from the lower chamber was removed 3 days later and used to evaluate the latency and bioactivity of TGF-β1 using enzyme-linked immunosorbent assay (ELISA) and mink lung (Mv1Lu) epithelial growth inhibition assay. Cells were also harvested and used for RNA extraction. The results of these experiments showed that 1) the TGF-β1-LAP complex, which was latent in traditionally used mink lung growth inhibition assay, directly modulated the expression of collagenase, type I, and type III collagen mRNA by dermal fibroblasts; 2) this stimulation was inhibited by M6P in a dose-dependent manner; 3) the TGF-β1-LAP inhibits Mv1Lu epithelial cells only when this complex was incubated with cell membranes isolated from dermal fibroblasts; and 4) LTGF-β1 activation seems to occur through a conformational alteration rather than by release of the mature TGF-β1 from LAP in our co-cultured system. This conformational alteration seems to occur through the interaction of the TGF-β1-LAP complex with the IGF-II/M6P receptors. Thus, the quantity of IGF-II/M6P receptors is important in cellular response to LTGF-β1 in any physiological and pathological conditions. J. Cell. Physiol. 180:61–70, 1999. © 1999 Wiley-Liss, Inc.