Pachymodulin, a New Functional Formyl Peptide Receptor 2 Peptidic Ligand Isolated from Frog Skin Has Janus-like Immunomodulatory Capacities

Recruitment of leukocytes is essential to fight infections or to heal injuries; however, excessive and/or prolonged responses favor the development of major inflammatory pathologies, such as cardiovascular or neurodegenerative diseases. Thus, it is of great interest to seek novel compounds that can regulate leukocyte recruitment depending on the degree of inflammation. We have isolated and characterized, by different chromatographic techniques, mass spectrometry, and Edman sequencing, a new hexapeptide (SSLSKL) from the Mexican frog <i>Pachymedusa dacnicolor,</i> which we named pachymodulin. In vitro, pachymodulin promotes the migration of leukocytes through the binding and activation of the human and mouse <i>N</i>-formyl peptide receptor 2 (huFPR2). In vivo, it exhibits opposite biological activities: under homeostatic conditions, pachymodulin induces the recruitment of leukocytes, whereas under inflammatory conditions, it inhibits this process. Therefore, pachymodulin represents an interesting template in the quest to design new immunomodulatory drugs in the therapy of immune-related diseases

CD43 Promotes Cells Transformation by Preventing Merlin-Mediated Contact Inhibition of Growth

<div><p>In normal tissues, strict control of tissue size is achieved by regulating cell numbers. The mechanism that controls total cell number is known as contact inhibition of growth and it depends on the NF2/Merlin pathway. Negative regulation of this pathway by deleterious mutations or by oncogenes results in cell transformation and tumor progression. Here we provide evidence that the CD43 sialomucin cooperates with oncogenic signals to promote cell transformation by abrogating the contact inhibition of growth through a molecular mechanism that involves AKT-dependent Merlin phosphorylation and degradation. Accordingly, inhibition of endogenous CD43 expression by RNA interference in lung, cervix and colon human cancer cells impaired tumor growth <i>in vivo</i>. These data underscore a previously unidentified role for CD43 in non-hematopoietic tumor progression. </p> </div

CD43 expression confers tumoral fitness to human tumor-derived cells.

<p>A549 lung (<b>A</b>), CasKi cervix (<b>B</b>) or DLD-1 colon (<b>C</b>) tumor cells containing the empty pSuper (pSup) vector or expressing the CD43 specific RNAi (RNAi) were cultured to confluence, the monolayer was then wounded and healing was evaluated (upper panel). Cells were also cultured in soft agar as indicated in material and methods, after three weeks colonies were counted (middle panel). Cells (1X10⁶ for A549, 3X10⁶ for CasKi or DLD-1) were injected subcutaneously into nu/nu mice; one month later, animals were sacrificed and tumor weight was evaluated (lower panel). Data represents the average ± SD of four independent experiments performed with four independent pSup or RNAi clones for each cell line. *p < 0.05, **p < 0.01 vs pSup.</p

Apoptosis in LCL and DCL patient lesions.

<p>Frozen tissue sections of LCL (A) and DCL (B) were stained with TUNEL. Two representative photographs are shown. C) Double staining of LCL patient tissue: TUNEL in peroxidase (brown), CD68 in phosphatase (red). Black arrows show TUNEL⁺ cells, red arrows show TUNEL⁺ CD68⁺ cells. D). Positive percentage analysis for TUNEL of 7 LCL and 5 DCL patients is shown. Three fields were counted, 200 cells per field (<i>p</i><0.05). Bar  = 20 µm.</p

Merlin mediates the inhibition of A549 cell proliferation by cell-cell contact.

<p><b>A</b>) A549 clones expressing the CD43 specific RNAi (RNAi) were transfected with non-specific (Ctl) or Merlin specific siRNAs (Merlin). 48 hrs after cells reached confluence total cell extract were prepared and Merlin protein levels (Merlin) as well as phosphorylated Yap levels (p-YAP) were evaluated by immunobloting using specific antibodies. Actin levels were used as loading control. <b>B</b>) The proliferation capacity of A549 clones expressing the CD43 specific RNAi (RNAi) transfected with non-specific (Ctl) or Merlin specific siRNA (Merlin) was evaluated at the indicated time points after cells reached confluence. Data represent the average of three independent experiments using two different clones.</p

IFNγ production by CD8 from LCL and DCL patients.

<p>A) PBMC were non-stimulated (CNT), stimulated with PMA-Ionomycin during 4 h or with MOi during 24 h. Density diagrams of IFNγ vs. CD8 from 10 LCL and 4 DCL patients are shown. B) CD8⁺IFNγ⁺ percentage analysis from 10 LCL and 4 DCL patients are shown. C) ELISA results of IFNγ production in supernatants of the co-incubation CD8-MOi from 9 LCL and 4 DCL patients are shown (<i>p</i><0.01).</p

CD43 signaling cooperates with oncogenic signals to promote cell transformation.

<p>In epithelial cells, upon interaction with putative ligand(s) present on the cell surface of neighboring cells or in the extracellular matrix, CD43 activates the PI3K/AKT pathway that results in the inhibition of the Hippo pathway by a mechanism involving Merlin phosphorylation and degradation, thus favoring cell survival and proliferation. Depending of the cellular context, signaling from intracellular CD43 located either on membrane vesicles or the nuclear membrane may also contribute to cellular transformation. Nonetheless, this might not be sufficient to overcome the anti-proliferative and death effects resulting from the activation of the ARF-p53 pathway also induced by CD43 [24]. However, in transformed cells with an impaired p53 pathway resulting from oncogenic signals like those provided by the E6 oncoprotein from HPV16, CD43 signaling promotes cells proliferation and tumor formation. </p

CD43 signaling cooperates with oncogenic signals to promote cell transformation.

<p>NIH-3T3-hEGFR (<b>A</b>) or E6/E7 transgenic mouse fibroblasts (<b>C</b>) carrying the pFNeo empty vector (pFNeo), expressing wild-type CD43 (Wt) or CD43 lacking the intracellular domain (ΔIC) were grown to confluence; the monolayer was then wounded (t=0) and healing was evaluated by light microscopy at the indicated time points. NIH-3T3-hEGFR fibroblasts were grown in soft agar as described in material and methods; after three weeks, colonies were counted (<b>B</b>). E6 transgenic mouse fibroblasts stably transfected with the indicated constructs were grown to confluence for three weeks; foci were stained with Giemsa and counted (<b>D</b>). 3X10⁶ E6/E7 fibroblasts stably transfected were injected subcutaneously into nu/nu mice; one month later, animals were sacrificed and tumor mass was weighed (<b>E</b>). Data shown are representative of at least four independent experiments performed with at least four independent pFNeo, Wt or ΔIC clones from each cell line. Graphs represent the average cell number ± SD of three independent experiments using at least 3 independent clones. *p < 0.05 vs pFNeo.</p

CD43 signaling targets the Merlin pathway.

<p>A549 clones containing the empty pSuper (pSup) vector or expressing the CD43 specific RNAi (RNAi) were grown to confluence (t=0) and further cultured for the indicated time points. At each time total cell extracts were prepared and the phosphorylation levels of STAT3 (p-STAT3), AKT (p-AKT), and GSK3β (p-GSK3) (<b>A</b>) as well as total Merlin levels (<b>B</b>) were determined by immunoblot, using specific antibodies. ERK protein levels were determined as loading control. <b>C</b>) Total cell extracts from A549 lung tumor cells cultured to confluence (t=0) or further cultured for 48 hrs in the absence (-) or presence of 20 μM LY294002 were resolved by SDS-PAGE and Merlin protein levels (Merlin) or phosphorylated AKT (pAKT) were evaluated by immunoblot with specific antibodies. ERK protein levels were used as loading control. <b>D</b>) A549 clones containing the empty pSuper (pSup) vector or expressing the CD43 specific RNAi (RNAi) were grown to confluence (t=0) and cultures were maintained for the indicated time points. Total cell extracts were prepared and the phosphorylation levels of YAP (p-YAP), ERK protein levels (loading control) were determined by immunoblot using specific antibodies. <b>E</b>) A549 clones expressing the empty pSuper (pSup) vector or the CD43 specific RNAi (RNAi) were grown to confluence and cultures were maintained in the absence or presence of 20 μM LY294002 (+LY) for 48 hrs. Cells were then harvested and counted. The graph represents the average cell number ± SD of three independent experiments using at least three independent clones. *p < 0.05, **p < 0.01 vs pSup.</p

Effector functions of CD8 from DCL patients were restored by pre-stimulation with TLR2 ligands.

<p>CD8 from DCL peripheral blood were purified and incubated in <i>L. mexicana</i> LPG [10 µg/ml] (CD8^L) or Pam3Cys [2 µg/ml] (CD8^P) for 24 h and washed twice. CD8 were analyzed for: A) Cytotoxicity, B) IFNγ production and C) Proliferation. One representative of 3 DCL patients is shown. D) Analysis of mean fluorescence intensity (MFI) for PD-1 expression on non-stimulated (CNT), LPG or Pam3Cys-stimulated CD8 from 3 DCL patients is shown (*<i>p</i> = 0.02, **<i>p</i> = 0.005).</p