Pluripotency gene expression and growth control in cultures of peripheral blood monocytes during their conversion into programmable cells of monocytic origin (PCMO): evidence for a regulatory role of autocrine activin and TGF-ß.

Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-ß(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TßRII, ALK5 as well as TGF-ß1 and the ßA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-ß1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-ß1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-ß signaling by either SB431542 or anti-TGF-ß antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-ß antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-ß/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-ß/Smad3, and to a lesser extent, activin/Smad2 signaling.

Rac1b negatively regulates TGF-ß1-induced cell motility in pancreatic ductal epithelial cells by suppressing Smad signalling.

Transforming growth factor (TGF)-ß1 promotes progression of pancreatic ductal adenocarcinoma (PDAC) by enhancing epithelial-mesenchymal transition, cell migration/invasion, and metastasis, in part by cooperating with the small GTPase Rac1. Prompted by the observation of higher expression of Rac1b, an alternatively spliced Rac1 isoform, in pancreatic ductal epithelial cells and in patients with chronic pancreatitis vs. PDAC, as well as in long-time vs. short-time survivors among PDAC patients, we asked whether Rac1b might negatively affect TGF-ß1 prometastatic function. Interestingly, the non-malignant pancreatic ductal epithelial cell line H6c7 exhibited a higher ratio of active Rac1b to total Rac1b than the TGF-ß1-responsive PDAC cell lines Panc-1 and Colo357. Notably, siRNA-mediated silencing of Rac1b increased TGF-ß1/Smad-dependent migratory activities in H6c7, Colo357, and Panc-1 cells, while ectopic overexpression of Rac1b in Panc-1 cells attenuated TGF-ß1-induced cell motility. Depletion of Rac1b in Panc-1 cells enhanced TGF-ß1/Smad-dependent expression of promoter-reporter genes and of the endogenous Slug gene. Moreover, Rac1b depletion resulted in a higher and more sustained C-terminal phosphorylation of Smad3 and Smad2, suggesting that Rac1b is involved in Smad2/3 dephosphorylation/inactivation. Since pharmacologic or siRNA-mediated inhibition of Smad3 but not Smad2 was able to alleviate the Rac1b siRNA effect on TGF-ß1-induced cell migration, our results suggests that Rac1b inhibits TGF-ß1-induced cell motility in pancreatic ductal epithelial cells by blocking the function of Smad3. Moreover, Rac1b may act as an endogenous inhibitor of Rac1 in TGF-ß1-mediated migration and possibly metastasis. Hence, it could be exploited for diagnostic/prognostic purposes or even therapeutically in late-stage PDAC as an antimetastatic agent.

Aberrant expression and secretion of heat shock protein 90 in patients with bullous pemphigoid.

The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses and its inhibition has proven successful in different mouse models of autoimmune diseases, including epidermolysis bullosa acquisita. Here, we investigated expression levels and secretory responses of Hsp90 in patients with bullous pemphigoid (BP), the most common subepidermal autoimmune blistering skin disease. In comparison to healthy controls, the following observations were made: (i) Hsp90 was highly expressed in the skin of BP patients, whereas its serum levels were decreased and inversely associated with IgG autoantibody levels against the NC16A immunodominant region of the BP180 autoantigen, (ii) in contrast, neither aberrant levels of circulating Hsp90 nor any correlation of this protein with serum autoantibodies was found in a control cohort of autoimmune bullous disease patients with pemphigus vulgaris, (iii) Hsp90 was highly expressed in and restrictedly released from peripheral blood mononuclear cells of BP patients, and (iv) Hsp90 was potently induced in and restrictedly secreted from human keratinocyte (HaCaT) cells by BP serum and isolated anti-BP180 NC16A IgG autoantibodies, respectively. Our results reveal an upregulated Hsp90 expression at the site of inflammation and an autoantibody-mediated dysregulation of the intracellular and extracellular distribution of this chaperone in BP patients. These findings suggest that Hsp90 may play a pathophysiological role and represent a novel potential treatment target in BP.

Pathogenicity of autoantibodies in anti-p200 pemphigoid.

Recently, the C-terminus of laminin γ1 has been identified as target antigen in anti-p200 pemphigoid and the disease was renamed as anti-laminin γ1 pemphigoid. However, the pathogenic relevance of these autoantibodies has not yet been demonstrated. Therefore, we employed an ex vivo model of autoantibody-mediated leukocyte-dependent neutrophil activation and dermal-epidermal separation (DES) using cryosections of human skin. We showed that anti-p200 pemphigoid sera (n = 7) induced DES in a time-dependent manner, in contrast to sera from healthy controls. Furthermore, laminin γ1-specific IgG and serum depleted from anti-laminin γ1 reactivity were generated using the recombinant C-terminus of laminin γ1 (LAMC1-term; amino acids 1364 to 1609). Interestingly, both fractions labeled the dermal-epidermal-junction (DEJ) by indirect immunofluorescence microscopy on human foreskin and recognized a 200 kDa protein by immunoblotting with dermal extract. Human and rabbit IgG against LAMC1-cterm failed to attract neutrophils at the DEJ and to induce DES. In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG. Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions. In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ. We conclude that autoantibodies in anti-p200 pemphigoid sera are pathogenic while pathogenicity is not mediated by autoantibodies against laminin γ1. Further studies are needed to identify the pathogenically relevant autoantigen in anti-p200 pemphigoid.

Enzymatic autoantibody glycan hydrolysis alleviates autoimmunity against type VII collagen.

Autoantibody-mediated diseases comprise a heterogeneous group of disorders in which the pathogenic potential of autoantibodies has been clearly demonstrated. In general, their treatment relies on the long-term use of systemic corticosteroids and other immunosuppressants that are associated with considerable adverse reactions. EndoS, an endoglycosidase derived from Streptococcus pyogenes, specifically hydrolyzes the N-linked glycan of native IgG and has previously been shown to modulate the interaction between the Fc portion of autoantibody and Fcγ receptors on leukocytes. Here, different models of autoimmunity to type VII collagen, a structural protein of the dermal-epidermal junction (DEJ), were employed to explore the therapeutic potential of EndoS. First, pretreatment of otherwise pathogenic anti-murine type VII collagen (mCOL7) IgG with EndoS significantly reduced split formation at the DEJ in cryosections of murine skin and abrogated clinical disease in mice. Next, the effect of EndoS was also seen when the enzyme was injected into mice after pathogenic anti-mCOL7 IgG had been administered. Finally, to mimic the patient situation even closer, EndoS was applied in mice that had already developed clinical disease after immunization with mCOL7. In all EndoS-treated mice, disease progression was stopped, and in the majority of mice, clinical disease even regressed. Of note, EndoS was shown to hydrolyze already in vivo-bound pathogenic autoantibodies. In addition, EndoS treatment decreased lesional expression of activating FcγRs while increasing FcγRIIB expression.

Differential roles of Smad2 and Smad3 in the regulation of TGF-ß1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1.

BACKGROUND: Progression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-ß)/Smad signalling pathway, eventually resulting in loss of TGF-ß-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-ß are mediated solely or partially through the canonical Smad signalling pathway which commences with activation of receptor-regulated Smads (R-Smads) Smad2 and Smad3 by the TGF-ß type I receptor. However, little is known on the relative contribution of each R-Smad, the possible existence of functional antagonism, or the crosstalk with other signalling pathways in the control of TGF-ß1-induced growth inhibition and cell migration. Using genetic and pharmacologic approaches we have inhibited in PDAC cells endogenous Smad2 and Smad3, as well as a potential regulator, the small GTPase Rac1, and have analysed the consequences for TGF-ß1-mediated growth inhibition and cell migration (chemokinesis). RESULTS: SiRNA-mediated silencing of Smad3 in the TGF-ß responsive PDAC cell line PANC-1 reduced TGF-ß1-induced growth inhibition but increased the migratory response, while silencing of Smad2 enhanced growth inhibition but decreased chemokinesis. Interestingly, siRNA-mediated silencing of the small GTPase Rac1, or ectopic expression of a dominant-negative Rac1 mutant largely mimicked the effect of Smad2 silencing on both TGF-ß1-induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-ß1-induction of a Smad2-specific transcriptional reporter and Smad2 C-terminal phosphorylation in PDAC cells while Smad3-specific transcriptional activity and Smad3 C-terminal phosphorylation appeared increased. Disruption of autocrine TGF-ß signalling in PANC-1 cells rendered cells less susceptible to the growth-suppressive effect of Rac1 inhibition, suggesting that the decrease in "basal" proliferation upon Rac1 inhibition was caused by potentiation of autocrine TGF-ß growth inhibition. CONCLUSIONS: In malignant cells with a functional TGF-ß signalling pathway Rac1 antagonizes the TGF-ß1 growth inhibitory response and enhances cell migration by antagonistically regulating Smad2 and Smad3 activation. This study reveals that Rac1 is prooncogenic in that it can alter TGF-ß signalling at the R-Smad level from a tumour-suppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression.

The Src family kinase inhibitors PP2 and PP1 block TGF-beta1-mediated cellular responses by direct and differential inhibition of type I and type II TGF-beta receptors.

Both the nonreceptor tyrosine kinase Src and the receptors for transforming growth factor (TGF)-ß (TßRI, TßRII) play major roles during tumorigenesis by regulating cell growth, migration/invasion and metastasis. The common Src family kinase inhibitors PP2 and PP1 effectively block Src activity in vitro and in vivo, however, they may exert non-specific effects on other kinases. In this study, we have evaluated PP2 and PP1 for their ability to inhibit TGFß1-mediated responses in the TGF-ß-responsive pancreatic adenocarcinoma cell line Panc1. We show that PP2 and PP1 but not the more specific Src inhibitor SU6656 effectively relieved TGF-b1-induced growth arrest and p21(WAF1) induction, while basal growth was enhanced by PP2 and PP1, and suppressed by SU6656. PP2 and PP1 but not SU6656 also suppressed TGF-ß1-induced epithelial-to-mesenchymal transition (EMT) as evidenced by their ability to inhibit downregulation of the epithelial marker E-cadherin, and upregulation of the EMT-associated transcription factor Slug. Likewise, PP2 and PP1 but not SU6656 effectively blocked TGF-ß1-induced activation of Smad2 and p38 MAPK and partially suppressed Smad activation and transcriptional activity on TGF-ß/Smad-responsive reporters of a kinase-active TßRI mutant ectopically expressed in Panc1 cells. Interestingly, PP2 and PP1 strongly inhibited recombinant TßRI in an in vitro kinase assay, with PP1 being more potent and PP2 being nearly as potent as the established TßRI inhibitor SB431542. PP2 but not PP1 also weakly inhibited the TßRII kinase. Together, these data provide evidence that PP2 and PP1 are powerful inhibitors of TßR function that can block TGF-ß/Smad signaling in a Src-unrelated fashion. Both agents may be useful as dual TGF-ß/Src inhibitors in experimental therapeutics of late stage metastatic disease.

Differential roles of Src in transforming growth factor-ß regulation of growth arrest, epithelial-to-mesenchymal transition and cell migration in pancreatic ductal adenocarcinoma cells.

Both transforming growth factor (TGF)-ß and the non-receptor tyrosine kinase Src play major roles during tumorigenesis by regulating cell growth, epithelial-to-mesenchymal transition (EMT), migration/invasion and metastasis, but little is known about the signaling crosstalk between them. To interfere with Src function in vitro and in vivo many studies have employed the pharmacologic Src inhibitors PP2 and PP1. Both agents have recently been shown to be powerful inhibitors of TGF-ß receptor type I/ALK5 and type II. As this situation prohibited any definite conclusions with respect to the relative contribution of TGF-ß vs. Src signaling, we decided to reappraise a potential role of Src in TGF-ß1-mediated cellular responses using RNA and dominant-negative (dn) interference to block Src expression and function, respectively. In TGF-ß-responsive pancreatic ductal adenocarcinoma (PDAC) cells, we show that Src is activated by TGF-ß1 and that its specific inhibition strongly attenuated basal proliferation and enhanced TGF-ß1-mediated growth arrest. However, Src inhibition was unable to impair TGF-ß1-controlled EMT as evidenced by cell morphology and regulation of the epithelial marker E-cadherin. Despite its dispensibility for TGF-ß-induced EMT, specific inhibition of Src dramatically reduced basal and TGF-ß1-induced cell migration in Panc-1 cells as measured with a novel real-time migration assay (xCELLigence DP system). Biochemically, dnSrc inhibition failed to block TGF-ß1/ALK5-induced activation of Smad2 and Smad3, but partially inhibited transcriptional activation of TGF-ß/Smad-responsive reporter genes, and effectively blocked basal and TGF-ß1-induced activation of p38 MAPK. Together, the data provide evidence for a role of Src in the regulation of basal proliferation as well as in basal and TGF-ß1-mediated cell motility but not EMT in TGF-ß-responsive pancreatic (tumor) cells.

The generation of programmable cells of monocytic origin involves partial repression of monocyte/macrophage markers and reactivation of pluripotency genes.

We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into hepatocyte-like cells using appropriate differentiation media. Phenotype conversion required prior in vitro culture in the presence of M-CSF, IL-3, and human serum, during which the cells acquired a state of plasticity, so were termed "programmable cells of monocytic origin" (PCMO). Here, we have further characterized the process of PCMO generation with respect to markers of monocyte-to-macrophage transition and pluripotency. During a 6-day culture period, various monocyte/macrophage differentiation markers were down-regulated being indicative of a process of partial dedifferentiation. Dedifferentiation and hepatic redifferentiation also proceeded in highly purified monocyte preparations, albeit with different kinetics, suggesting that the presence of nonmonocytes, or soluble factors derived from them, is not essential in order for monocytes to acquire a multipotent state. PCMOs expressed various markers of human embryonic stem cells with early induction of NANOG and OCT4. Expression of the pluripotency-associated OCT4A isoform was paralleled by a global rise in histone H3 methylation on Lys-4, a marker of active chromatin, and coincided with peak sensitivity to tissue-specific differentiation. These results show that peripheral blood monocytes can be induced in vitro to transiently acquire stem cell-like properties and concomitantly a state of increased differentiation potential toward the hepatocytic phenotype.

Antitumor activity of ALK1 in pancreatic carcinoma cells.

In this study, the authors investigated the expression of activin receptor-like kinase 1 (ALK1) in pancreatic carcinoma and evaluated its potential role as a tumor suppressor in vitro and in vivo. Endogenous ALK1 expression was demonstrated by immunohistochemistry in both pancreatic tumor tissue and peritumoral normal tissue from 6 patients and by RT-PCR in 8/12 established pancreatic cancer cell lines. Ectopic expression of a constitutively active (ca) ALK1 mutant in TGF-beta sensitive PANC-1 and COLO-357 cells augmented transcriptional activation of a Smad2/3 responsive reporter, and slowed down basal growth in vitro. Both effects were further enhanced by TGF-beta/ALK5 stimulation, suggesting largely independent nuclear Smad signaling by both type I receptors. Upon orthotopic transplantation of PANC-1-caALK1 into immunodeficient mice, tumor size was strongly reduced and was associated with a lower microvessel density in the PANC-1-caALK1-derived tumors. In vitro, this mutant efficiently blocked TGF-beta-induced epithelial-to-mesenchymal transdifferentiation and suppressed TGF-beta/ALK5-mediated activation of the p38 MAPK pathway. Mechanistically, caALK1 silenced MyD118, an immediate TGF-beta target gene whose protein product, GADD45beta, couples Smad signaling to p38 activation. These results show that ALK1 activation in pancreatic tumor cells is antioncogenic by inducing ALK5-independent growth inhibition and by blocking TGF-beta/ALK5-mediated epithelial-to-mesenchymal transdifferentiation and, possibly, invasion and metastatic progression.

Adhesion and Rac1-dependent regulation of biglycan gene expression by transforming growth factor-beta. Evidence for oxidative signaling through NADPH oxidase.

Both transforming growth factor-beta (TGF-beta)-induced expression of biglycan (BGN) and activation of p38 MAPK have been implicated in cellular adhesion and migration. Here, we analyzed the role of adhesive events and the small GTPase Rac1 in TGF-beta regulation of BGN. TGF-beta1 induction of BGN expression and activation of p38 was abolished or strongly reduced when cells were kept in suspension or exposed to either the actin cytoskeleton-disrupting agent cytochalasin D or a specific chemical Rac1 inhibitor. Ectopic expression of a dominant negative mutant (T17N) of Rac1 abrogated both TGF-beta-induced p38 MAPK activation and BGN up-regulation but did not affect TGF-beta-induced phosphorylation of Smad3 or transcriptional induction of Growth Arrest DNA Damage 45beta, previously shown to be crucial for TGF-beta regulation of BGN. Overexpression of wild type Rac1 greatly enhanced the TGF-beta effect on BGN in adherent cells, whereas ectopic expression of constitutively active Rac1 (Q61L) activated p38 and in the presence of exogenous TGF-beta was able to rescue BGN expression in nonadherent cells. Endogenous Rac1 was activated by TGF-beta treatment in PANC-1 cells in an adhesion-dependent fashion. Like Rac1-T17N, the NADPH oxidase inhibitor diphenylene iodonium and the tyrosine kinase inhibitor herbimycin A blocked TGF-beta-induced p38 activation and BGN expression, suggesting that Rac1 exerts its effect on BGN and p38 through increasing NADPH oxidase activity and subsequent production of reactive oxygen species. These results show that the TGF-beta effect on BGN is dependent on cell adhesion and that activated Rac1, presumably acting through NADPH oxidase(s), is necessary but not sufficient for TGF-beta-induced BGN expression.

Transforming growth factor-beta (TGF-beta) type I receptor/ALK5-dependent activation of the GADD45beta gene mediates the induction of biglycan expression by TGF-beta.

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.