Dihydrocytochalasin B Enhances Transforming Growth Factor-β-Induced Reexpression of the Differentiated Chondrocyte Phenotype without Stimulation of Collagen Synthesis

Rabbit articular chondrocytes were treated with retinoic acid (RA) to eliminate the differentiated phenotype marked by the synthesis of type II collagen and high levels of proteoglycan. Exposure of such cells to transforming growth factor-β1 (TGF-β1) in secondary culture under serum-free and RA-free, defined conditions led to reexpression of the differentiated phenotype. The microfilament modifying drug, dihydrocytochalasin B (DHCB), enhanced the effectiveness of TGF-β1 and produced a threefold stimulation of type II collagen reexpression (measured by 2-D CNBr peptide mapping) at 0.3 ng/ml TGF-β1 without altering total collagen synthesis. Type II collagen reexpression was maximal from 1 to 5 ng/ml TGF-β1, with or without DHCB. The effect of DHCB on proteoglycan synthesis was maximal at 1 ng/ml TGF-β1. At this dose TGF-β alone produced no increase in ³⁵ SO₄ incorporation, while simultaneous treatment with DHCB caused a sevenfold stimulation of proteoglycan synthesis. DHCB-independent stimulation of proteoglycan reexpression occurred between 5 and 15 ng/ml TGF-β1. In contrast, TGF-β1-dependent stimulation of proteoglycan synthesis in differentiated chondrocytes in primary monolayer culture was not substantially affected by DHCB. The collagen data suggest that TGF-β1 utilizes separate pathways to control phenotypic change and collagen (matrix) synthesis. Microfilament modification by DHCB selectively enhances the effectiveness of the TGF-β1-dependent signaling pathway that controls reexpression of the differentiated phenotype.     

Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

Collagen biosynthesis by human skin fibroblasts. III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. the results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased the amount of [3H]hydroxyproline synthesized. Ascorbic acid, however, did not increase the synthesis of 3H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and secretion of procollagen.     

Differential Effects of TGF-β1 on Hyaluronan Synthesis by Fetal and Adult Skin Fibroblasts: Implications for Cell Migration and Wound Healing

Fetal wound healing differs from its adult counterpart in that it is regenerative and occurs without scarring. The matrix macromolecule hyaluronan (HA) and various cytokines, including members of the TGF-β family, have been implicated in the control of scarring. We have previously reported that adult and fetal fibroblasts differ with respect to the effect of cell density on HA synthesis when cultured on plastic tissue culture dishes. Data regarding the effects of substratum and TGF-β1 on HA synthesis by these cells are presented in this communication. Our results indicate that HA synthesis by both fetal and adult fibroblasts is (a) up-regulated by culture on a collagen substratum and (b) differentially regulated by TGF-β1 in a manner which is dependent upon both substratum and cell density. TGF-β1 stimulated HA synthesis by confluent fetal fibroblasts growing on a plastic substratum, but inhibited HA synthesis on a collagen substratum; these data underscore the important role of the substratum in determining the precise effect of TGF-β1 on cell behavior. Related studies indicated that the migration of fetal and adult fibroblasts into the collagen substrata was modulated by TGF-β1 in a manner identical to its effect on HA synthesis. These observations are discussed in terms of the contribution of distinct fibroblast subpopulations to wound healing and the manner in which this is regulated by matrix and cytokines.     

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.     

Asiatic Acid Isolated From Centella Asiatica Inhibits TGF-β1-induced Collagen Expression in Human Keloid Fibroblasts via PPAR-γ Activation

Keloids are fibroproliferative disorders characterized by exuberant extracellular matrix deposition and transforming growth factor (TGF)-β/Smad pathway plays a pivotal role in keloid pathogenesis. Centella asiatica extract has been applied in scar management for ages. As one of its major components, asiatic acid (AA) has been recently reported to inhibit liver fibrosis by blocking TGF-β/Smad pathway. However, its effect on keloid remains unknown. In order to investigate the effects of AA on cell proliferation, invasion and collagen synthesis, normal and keloid fibroblasts were exposed to TGF-β1 with or without AA. Relevant experiments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, Transwell invasion assay, enzyme-linked immunosorbent assay, Western blot, quantitative polymerase chain reaction and RNA interference assay were conducted. As a result, keloid fibroblasts showed higher responsiveness to TGF-β1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-β1-induced expression of collagen type I, inhibit Smad 2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) expression, while elevate Smad 7 protein level. Noteworthy, the effects of AA on keloid fibroblasts could be abrogated by PPAR-γ antagonist GW9662 and by silencing of PPAR-γ. The present study demonstrated that AA inhibited TGF-β1-induced collagen and PAI-1 expression in keloid fibroblasts through PPAR-γ activation, which suggested that AA was one of the active constituents of C. asiatica responsible for keloid management, and could be included in the arsenal for combating against keloid.     

The regulation of aggrecanase ADAMTS-4 expression in human Achilles tendon and tendon-derived cells

Several members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family have been identified as aggrecanases, whose substrates include versican, the principal large proteoglycan in the tendon extracellular matrix. We have characterized the expression of ADAMTS-4 in human Achilles tendon and tendon-derived cells. ADAMTS-4 mRNA levels were higher in ruptured tendon compared with normal tendon or chronic painful tendinopathy. In tissue extracts probed by Western blotting, mature ADAMTS-4 (68 kDa) was detected only in ruptured tendons, while processed ADAMTS-4 (53 kDa) was detected also in chronic painful tendinopathy and in normal tendon. In cultured Achilles tendon cells, transforming growth factor-β (TGF-β) stimulated ADAMTS-4 mRNA expression (typically 20-fold after 24 h), while interleukin-1 induced a smaller, shorter-term stimulation which synergised markedly with that induced by TGF-β. Increased levels of immunoreactive proteins consistent with mature and processed forms of ADAMTS-4 were detected in TGF-β-stimulated cells. ADAMTS-4 mRNA was expressed at higher levels by tendon cells in collagen gels than in monolayer cultures. In contrast, the expression of ADAMTS-1 and -5 mRNA was lower in collagen gels compared with monolayers, and these mRNA showed smaller or opposite responses to growth factors and cytokines compared with that of ADAMTS-4 mRNA. We conclude that both ADAMTS-4 mRNA and ADAMTS-4 protein processing may be differentially regulated in normal and damaged tendons and that both the matrix environment and growth factors such as TGF-β are potentially important factors controlling ADAMTS aggrecanase activities in tendon pathology.     

Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-β1-Induced Fibrotic Phenotype in Lung Fibroblasts

Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-β1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-β-D-xylopyranoside, a competitive inhibitor of β4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-β1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-β1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of β4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of β4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis.     

α2β1 Integrin Mediates Dermal Fibroblast Attachment to Type VII Collagen via a 158-Amino-Acid Segment of the NC1 Domain

Dermal fibroblasts are in apposition to type VII (anchoring fibril) collagen in both unwounded and wounded skin. The NC1 domain of type VII collagen contains multiple submodules with homology to known adhesive molecules, including fibronectin type III-like repeats and a potential RGD cell attachment site. We previously reported the structure and matrix binding properties of authentic and recombinant NC1. In this study, we examined the interaction between dermal fibroblasts and the NC1 domain of type VII collagen. We found that both recombinant and authentic NC1 vigorously promoted human fibroblast attachment. Adhesion of fibroblasts to NC1 was dose dependent, saturable, and abolished by both polyclonal and monoclonal antibodies to NC1. Cell adhesion to NC1 was divalent cation dependent and specifically inhibited by a monoclonal antibody directed against the α2 or β1 integrin subunits, but not by the presence of RGD peptides. Furthermore, the cell-binding activity of NC1 was not conformation dependent, since heat-denatured NC1 still promoted cell adhesion. Using a series of recombinant NC1 deletion mutant proteins, the cell binding site of NC1 was mapped to a 158-aa (residues 202–360) subdomain. We conclude that human dermal fibroblasts interact with the NC1 domain of type VII collagen and this cell–matrix interaction is mediated by the α2β1 integrin and is RGD independent.     

Culture of human dermal fibroblasts in collagen gels: modulation of interleukin 1-induced prostaglandin E2 synthesis by an extracellular matrix.

Human dermal fibroblasts, cultured as suspensions in collagen gels and as monolayers, were stimulated with recombinant human interleukin-1 beta (rIL 1 beta) at 72 h, and prostaglandin E2 (PGE2) was assayed 24 h later. Fibroblasts in gels were less responsive to rIL 1 beta than monolayers, PGE2 synthesis increasing from less than 1 ng/microgram DNA without rIL 1 beta to maxima of 11.3 and 32.9 ng/micrograms DNA, with half maximal release occurring at 7.47 and 0.75 pM rIL 1 beta for the gel and monolayer cultures, respectively. Increased PGE2 was first detected 4 h after addition of rIL 1 beta to gels and was inhibited by 10(-5) M indomethacin. The amount of PGE2 synthesized per fibroblast increased with the time the gels had been in culture when stimulated with rIL 1 beta and was proportional to the number of fibroblasts in the gels, but inversely related to the collagen concentration. A common feature of these experiments was significantly greater induction of PGE2 synthesis at higher cell densities in collagen gels. Exogenous 10(-4) M arachidonic acid further increased PGE2 synthesis by rIL 1 beta-stimulated fibroblasts, but the differential in the amount of PGE2 released from fibroblasts at high and low population densities in the gels was maintained. These results are consistent with interleukin 1 (IL 1) stimulating PGE2 synthesis in dermal fibroblasts by increasing cyclooxygenase activity. Furthermore, the results show that dermal fibroblasts have an additional regulatory mechanism, related to the cell population densities or their interactions with an extracellular matrix, to finely modulate the amount of PGE2 synthesized in response to IL 1.     

B lymphocytes and B-cell activating factor promote collagen and profibrotic markers expression by dermal fibroblasts in systemic sclerosis

Introduction

B lymphocytes might play a pathogenic role in dermal fibrosis in systemic sclerosis (SSc). B-cell activating factor (BAFF), a key cytokine for B-cell activation, is increased in the serum and the skin of patients with SSc. However, the ability of B cells directly to stimulate dermal fibroblasts and the role of BAFF are not fully understood. We therefore investigated the involvement of B cells and BAFF in the expression of collagen and profibrotic markers by dermal fibroblasts.

Methods

Cocultures of blood B cells from healthy blood donors and normal or SSc dermal fibroblasts stimulated with anti-IgM and BAFF were performed. Alpha-SMA, TIMP1, MMP9, COL1A1, COL1A2, and COL3A1 mRNA expression were determined by quantitative RT-PCR. Soluble collagen, BAFF, IL-6, IL-1β, TGF-β1, and CCL2 protein secretion were assessed.

Results

Coculture of blood B cells and dermal fibroblasts isolated from SSc patients induced IL-6, TGF-β1, CCL2, and collagen secretion, as well as Alpha-SMA, TIMP1, and MMP9 expression in dermal fibroblasts. Transwell assays demonstrated that this induction was dependent on cell-cell contact. Addition of anti-IgM and BAFF to the coculture increased IL-6, CCL2, TGF-β1, and collagen secretion. B cell- and BAFF-induced collagen secretion was highly reduced by anti-TGF-β1 antibodies.

Conclusions

Our results showed for the first time a direct role of B cells on the production of collagen by dermal fibroblasts, which is further enhanced by BAFF. Thus, these results demonstrate a new pathogenic role of B cells and BAFF in fibrosis and systemic sclerosis.     

Hydrogen Sulfide Alleviates Myocardial Collagen Remodeling in Association with Inhibition of TGF-β/Smad Signaling Pathway in Spontaneously Hypertensive Rats

The study was designed to explore the role and possible mechanisms of hydrogen sulfide (H₂S) in the regulation of myocardial collagen remodeling in spontaneously hypertensive rats (SHRs). We treated nine-week-old male SHRs and age- and sex-matched Wistar–Kyoto rats (WKYs) with NaHS (90 μmol/kg⁻¹·day⁻¹) for 9 wks. At 18 wks, plasma H₂S, tail arterial pressure, morphology of the heart, myocardial ultrastructure and collagen volume fraction (CVF), myocardial expressions of collagen I and III protein and procollagen I and III mRNA, transforming growth factor-β1 (TGF-β1), TGF-β type I receptor (TβR-I), type II receptor (TβR-II), p-Smad2 and 3, matrix metalloproteinase (MMP)-13 and tissue inhibitors of MMP (TIMP)-1 proteins were determined. TGF-β1-stimulated cultured cardiac fibroblasts (CFs) were used to further study the mechanisms. The results showed that compared with WKYs, SHRs showed a reduced plasma H₂S, elevated tail artery pressure and increased myocardial collagen, TGF-β1, TβR-II, p-Smad2 and p-Smad3 expressions. However, NaHS markedly decreased tail artery pressure and inhibited myocardial collagen, TGF-β1, TβR-II, p-Smad2 and p-Smad3 protein expressions, but H₂S had no effect on the expressions of MMP-13 and TIMP-1. Hydralazine reduced blood pressure but had no effect on myocardial collagen, MMP-13 and TIMP-1 expressions and TGF-β1/Smad signaling pathway. H₂S prevented activation of the TGF-β1/Smad signaling pathway and abnormal collagen synthesis in CFs. In conclusion, the results suggested that H₂S could prevent myocardial collagen remodeling in SHR. The mechanism might be associated with inhibition of collagen synthesis via TGF-β1/Smad signaling pathway.     

A member of the Y-box protein family interacts with an upstream element in the α1(I) collagen gene

Transforming growth factor beta (TGF-β) stimulates protein complex formation on a TGF-β response element (TAE) found in the distal portion (−1624) of the collagen alpha 1(I) promoter. To identify the fibroblast proteins in this complex, an expression library constructed from human embryonic lung fibroblasts mRNA was screened using a tetramer of TAE. Y-box binding protein (YB-1), was identified as a protein in the TAE–protein complex. The protein expressed by phage clones formed a specific complex with labeled TAE but not mutated TAE (mTAE) similar to the complex formed with nuclear protein. Nuclear protein–TAE complexes isolated from native gels contained YB-1 by Western analysis. TGF-β treatment increased the amount of YB-1 protein in nuclear extracts, decreased its amount in cytoplasm, but did not alter the steady state levels of YB-1 mRNA. A full-length YB-1 protein expressed in human lung fibroblasts was primarily located in the nucleus with punctate staining in cytoplasmic regions. The expression of YB-1 decreased in the cytoplasm after 2 h of TGF-β treatment. Therefore, the increased binding activity seen in TGF-β-stimulated nuclear extracts was due primarily to relocalization of YB-1 from the cytoplasm to the nuclear compartment. Co-transfection of YB-1 cDNA with a collagen promoter–reporter construct caused a dose-dependent activation of collagen promoter activity in rat fibroblasts whereas the promoter with a mutation in the TAE element was not sensitive to YB-1 co-expression. In conclusion, we have identified YB-1 as a protein that interacts with a TGF-β response element in the distal region of the collagen alpha 1(I) gene. YB-1 protein activates the collagen promoter and translocates into the nucleus during TGF-β addition to fibroblasts, suggesting a role for this protein in TGF-β signaling.     

TGF-β1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-β1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-β1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-β1 and serum significantly augmented collagen gel contraction. TGF-β1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-β1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-β induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.     

The Growth-Inhibitory Block of TGF-β Is Located Close to the G1/S Border in the Cell Cycle

Transforming growth factor-β (TGF-β) inhibits DNA synthesis in dense cultures of young human embryonic fibroblasts and antagonizes the mitogenic action of platelet-derived growth factor (PDGF). The inhibition of the PDGF-BB action by TGF-β was independent of the induction of mRNAs for the PDGF-A chain and PDGF-β receptor, the predominant types of PDGF receptor in human fibroblasts. The TGF-β-mediated inhibition did not influence the expression of various genes that are involved in the transition from the arrested (G0) state to the S phase of the cell cycle. Indeed, TGF-β upregulated the "early" genes c-myc, c-fos, and jun B and downregulated the growth arrest-specific (gas) genes. These results suggest that the inhibition of DNA synthesis by TGF-β in human fibroblasts is independent of modulation of expression of early and gas genes, placing the TGF-β block comparatively late in the G0 to S transition. In cultures of senescent human fibroblasts TGF-β stimulated DNA synthesis but, nevertheless, had the same effect as in young cells on the expression of PDGF chains and receptor genes, as well as on early and gas genes, with the exception of a significantly lower induction of c-fos.     

A Three-dimensional Collagen Lattice Induces Protein Kinase C-ζ Activity: Role in α2 Integrin and Collagenase mRNA Expression

A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment that simulates normal dermis. Such a collagen matrix environment regulates interstitial collagenase (type I metalloproteinase [MMP-1], collagenase-1) and collagen receptor α₂ subunit mRNA expression in both unstimulated or platelet-derived growth factor–stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239–249). Here we report that the collagen gel can signal protein kinase C (PKC)-ζ activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-ζ immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-ζ protein levels or intracellular location was observed. DNA binding activity of nuclear factor κB (NF-κB), a downstream regulatory target of PKC-ζ, was also increased by fibroblasts grown in collagen gel. The composition of the NF-κB/Rel complexes that contained p50, was not changed. The potential role of PKC-ζ in collagen gel–induced mRNA expression of collagen receptor α₂ subunit and human fibroblast MMP-1 was assessed by the following evidence. Increased levels of α₂ and MMP-1 mRNA in collagen gel–stimulated fibroblasts were abrogated by bisindolylmaleimide GF 109203X and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)–inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5′ end of PKC-ζ mRNA sequences significantly reduced the collagen lattice–stimulated α₂ and MMP-1 mRNA levels. Taken together, these data indicate that PKC-ζ, a PKC isoform not inducible by PMA or diacylglycerol, is a component of collagen matrix stimulatory pathway for α₂ and MMP-1 mRNA expression. Thus, a three-dimensional collagen lattice maintains the dermal fibroblast phenotype, in part, through the activation of PKC-ζ.