Limits...
The Urokinase/Urokinase Receptor System in Mast Cells: Effects of its Functional Interaction with fMLF Receptors

View Article: PubMed Central - PubMed

ABSTRACT

Mast cell and basophils express the high affinity receptor for IgE (FcɛRI) and are primary effector cells of allergic disorders. The urokinase (uPA)-mediated plasminogen activation system is involved in physiological and pathological events based on cell migration and tissue remodelling, such as inflammation, wound healing, angiogenesis and metastasis. uPA is a serine protease that binds uPAR, a high affinity glycosyl-phosphatidyl-inositol (GPI)-anchored receptor. uPAR focuses uPA activity at the cell surface and activates intracellular signaling through lateral interactions with integrins, receptor tyrosine kinases and the G-protein-coupled family of fMLF chemotaxis receptors (FPRs).

We investigated the expression of the uPA-uPAR system and its functional interaction with FPRs in human mast cells (MCs). Differently from basophils, MCs produced uPA that was able to induce their chemotaxis. Indeed, MCs also expressed uPAR, both in the intact and in a cleaved form (DII-DIII-uPAR) that can expose, at the N-terminus, the SRSRY sequence, able to interact with FPRs and to mediate cell chemotaxis. MCs also expressed mRNAs for FPRs that were functionally active; indeed, uPA and a soluble peptide (uPAR84–95), containing the SRSRY chemotactic sequence of uPAR and able to interact with FPRs, were able to induce MCs chemotaxis.

Thus, uPA is a potent chemoattractant for MCs acting through the exposure of the chemotactic epitope of uPAR, that is an endogenous ligand for FPRs. The same mechanism could be involved in VEGF-A secretion by human MCs, also induced by uPA and uPAR84–95 stimulation.

No MeSH data available.


Related in: MedlinePlus

uPA, uPAR and FPRs expression in HMC-1 cells.A: mRNA expression of uPA and uPAR in HMC-1 cells. Total RNA of THP-1 monocyte-like cells as a positive control (lane 1) and HMC-1 cells (lane 2) were prepared, reverse transcribed, and amplified by 40 PCR cycles in the presence of uPA and uPAR-specific primers and GAPDH primers, as a loading control. PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under UV illumination.B: Western blot analysis of uPA and uPAR in HMC-1 cells. THP-1 monocyte-like cells as a positive control (lane 1) and HMC-1 cells (lane 2) were lysed in Triton X-100 and 50 μg of total protein were analyzed by 9% SDS-PAGE and Western blot with an anti-uPA and anti-uPAR polyclonal antibody.C: mRNA expression of FPRs in HMC-1 cells. Total RNA was prepared, reverse transcribed and amplified by 40 PCR cycles in the presence of FPR1- (lane 1), FPR2- (lane 2), and FPR3- (lane 3) specific primers and GAPDH (lane 4) primers, as loading control. PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under UV illumination.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120748&req=5

f1-tm-15-34: uPA, uPAR and FPRs expression in HMC-1 cells.A: mRNA expression of uPA and uPAR in HMC-1 cells. Total RNA of THP-1 monocyte-like cells as a positive control (lane 1) and HMC-1 cells (lane 2) were prepared, reverse transcribed, and amplified by 40 PCR cycles in the presence of uPA and uPAR-specific primers and GAPDH primers, as a loading control. PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under UV illumination.B: Western blot analysis of uPA and uPAR in HMC-1 cells. THP-1 monocyte-like cells as a positive control (lane 1) and HMC-1 cells (lane 2) were lysed in Triton X-100 and 50 μg of total protein were analyzed by 9% SDS-PAGE and Western blot with an anti-uPA and anti-uPAR polyclonal antibody.C: mRNA expression of FPRs in HMC-1 cells. Total RNA was prepared, reverse transcribed and amplified by 40 PCR cycles in the presence of FPR1- (lane 1), FPR2- (lane 2), and FPR3- (lane 3) specific primers and GAPDH (lane 4) primers, as loading control. PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under UV illumination.

Mentions: To investigate the existence of a functional interaction between the uPA-uPAR system and FPRs in MCs, we first evaluated the expression at mRNA and protein level of uPA and uPAR in HMC-1 cells. uPA and uPAR mRNAs were detected by RT-PCR analysis of RNAs from HMC-1 cells and THP-1 monocyte-like cells, used as a positive control (Fig. 1A).


The Urokinase/Urokinase Receptor System in Mast Cells: Effects of its Functional Interaction with fMLF Receptors
uPA, uPAR and FPRs expression in HMC-1 cells.A: mRNA expression of uPA and uPAR in HMC-1 cells. Total RNA of THP-1 monocyte-like cells as a positive control (lane 1) and HMC-1 cells (lane 2) were prepared, reverse transcribed, and amplified by 40 PCR cycles in the presence of uPA and uPAR-specific primers and GAPDH primers, as a loading control. PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under UV illumination.B: Western blot analysis of uPA and uPAR in HMC-1 cells. THP-1 monocyte-like cells as a positive control (lane 1) and HMC-1 cells (lane 2) were lysed in Triton X-100 and 50 μg of total protein were analyzed by 9% SDS-PAGE and Western blot with an anti-uPA and anti-uPAR polyclonal antibody.C: mRNA expression of FPRs in HMC-1 cells. Total RNA was prepared, reverse transcribed and amplified by 40 PCR cycles in the presence of FPR1- (lane 1), FPR2- (lane 2), and FPR3- (lane 3) specific primers and GAPDH (lane 4) primers, as loading control. PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under UV illumination.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120748&req=5

f1-tm-15-34: uPA, uPAR and FPRs expression in HMC-1 cells.A: mRNA expression of uPA and uPAR in HMC-1 cells. Total RNA of THP-1 monocyte-like cells as a positive control (lane 1) and HMC-1 cells (lane 2) were prepared, reverse transcribed, and amplified by 40 PCR cycles in the presence of uPA and uPAR-specific primers and GAPDH primers, as a loading control. PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under UV illumination.B: Western blot analysis of uPA and uPAR in HMC-1 cells. THP-1 monocyte-like cells as a positive control (lane 1) and HMC-1 cells (lane 2) were lysed in Triton X-100 and 50 μg of total protein were analyzed by 9% SDS-PAGE and Western blot with an anti-uPA and anti-uPAR polyclonal antibody.C: mRNA expression of FPRs in HMC-1 cells. Total RNA was prepared, reverse transcribed and amplified by 40 PCR cycles in the presence of FPR1- (lane 1), FPR2- (lane 2), and FPR3- (lane 3) specific primers and GAPDH (lane 4) primers, as loading control. PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under UV illumination.
Mentions: To investigate the existence of a functional interaction between the uPA-uPAR system and FPRs in MCs, we first evaluated the expression at mRNA and protein level of uPA and uPAR in HMC-1 cells. uPA and uPAR mRNAs were detected by RT-PCR analysis of RNAs from HMC-1 cells and THP-1 monocyte-like cells, used as a positive control (Fig. 1A).

View Article: PubMed Central - PubMed

ABSTRACT

Mast cell and basophils express the high affinity receptor for IgE (FcɛRI) and are primary effector cells of allergic disorders. The urokinase (uPA)-mediated plasminogen activation system is involved in physiological and pathological events based on cell migration and tissue remodelling, such as inflammation, wound healing, angiogenesis and metastasis. uPA is a serine protease that binds uPAR, a high affinity glycosyl-phosphatidyl-inositol (GPI)-anchored receptor. uPAR focuses uPA activity at the cell surface and activates intracellular signaling through lateral interactions with integrins, receptor tyrosine kinases and the G-protein-coupled family of fMLF chemotaxis receptors (FPRs).

We investigated the expression of the uPA-uPAR system and its functional interaction with FPRs in human mast cells (MCs). Differently from basophils, MCs produced uPA that was able to induce their chemotaxis. Indeed, MCs also expressed uPAR, both in the intact and in a cleaved form (DII-DIII-uPAR) that can expose, at the N-terminus, the SRSRY sequence, able to interact with FPRs and to mediate cell chemotaxis. MCs also expressed mRNAs for FPRs that were functionally active; indeed, uPA and a soluble peptide (uPAR84–95), containing the SRSRY chemotactic sequence of uPAR and able to interact with FPRs, were able to induce MCs chemotaxis.

Thus, uPA is a potent chemoattractant for MCs acting through the exposure of the chemotactic epitope of uPAR, that is an endogenous ligand for FPRs. The same mechanism could be involved in VEGF-A secretion by human MCs, also induced by uPA and uPAR84–95 stimulation.

No MeSH data available.


Related in: MedlinePlus