β -Hexosaminidase–Induced Activation of p44/42 Mitogen-Activated Protein Kinase Is Dependent on p21Ras and Protein Kinase C and Mediates Bovine Airway Smooth-Muscle Proliferation

Abstract

Late-phase and sustained activation of p44/42(MAPK) has been reported to be a critical factor in cell mitogenesis. We therefore hypothesized that p44/42(MAPK) is involved in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth-muscle cells (ASMC). Treatment of adherent ASMC with beta-hexosaminidase A (Hex A, 50 nM), an endogenous mannosyl-rich glycoprotein, resulted in a late-onset (30-min) activation of p44/42(MAPK) that lasted for 4 h. Activation of p44/42(MAPK) induced by Hex A was inhibited by an 18-mer phosphorothioate-derivatized antisense oligonucleotide (1-5 microM) directed to human p44(MAPK); the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059 (5 microM); the p42(MAPK) inhibitor Tyrphostin AG-126 (0.2 microM); the farnesyl transferase inhibitors SCH-56582 (10 microM) and FPT III (10 miroM), which inhibit p21Ras activation; and Calphostin C (0.2 microM), an inhibitor of protein kinase C. These agents also inhibited Hex A-induced cell proliferation in bovine ASMC. These data suggest that Hex A activates p44/42(MAPK) in a p21Ras- and PKC-dependent manner and that this activation mediates Hex A- induced mitogenesis in bovine ASMC.

Isoenzymes A and B of N-acetyl-β-D-hexosaminidase in tissue of colon cancer

“…Zdaniem Lewa i wsp. [13] HEX A działający na kwaśne substraty, takie jak glikozoaminoglikany i glikolipidy oraz glikoproteiny zawierające kwasy sialowe, jest mediatorem stanu zapalnego wydzielanym do przestrzeni zewnątrz-komórkowej, odzwierciedla w płynach ustrojowych aktywność metaboliczną i wydzielniczą komórek. HEX B działający na obojętne łańcuchy oligosacharydowe jest wskaźnikiem uszkodzenia komórek [13].…”

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma

“…Finally, LEW et al [104] have demonstrated that induction of proliferation in bovine tracheal smooth muscle by bhexosaminidase and activation of the ERK pathway is prevented by the PKC inhibitor calphostin C, an effect which appears to be mediated at the Ras/Raf-1 locus ( fig. 3) [128].…”

Enzymic methylation of arginyl residues in -Gly-Arg-Gly- peptides

“…The derivatized sample was then injected onto a C ") reverse-phase column (Shodex C ") , 5 µm spherical particles, 4.6 mm internal diam.i25 cm) equilibrated in solvent A at 43 mC, and eluted at a flow rate of 1. Airway smooth-muscle cell culture Primary cultures of bovine tracheal smooth-muscle cells were prepared from bovine trachealis muscle, as described previously [21]. Direct immunofluorescence staining of the cells with an FITC-conjugated monoclonal antibody against smooth-muscle isoactin (Sigma) demonstrated that cultures consisted of a homogeneous population of smooth-muscle cells [22].…”

“…After a 48 h starvation, the inhibitors to be tested were added 30 min before stimulation of the cells. Without a change of medium, purified hexosaminidase A (Hex A) or vehicle (PBS) was added [21]. In some experiments, an agonistic antibody (mAb "& ; [23]) directed to the extracellular domain of human macrophage mannose receptors was used to stimulate cells.…”

“…Parallel experiments were set up to assess cell counts and cytotoxicity by tetrazolium salt reduction with the use of a 3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyl tetrazolium bromide (5 mg\ ml ; Millipore-filtered) (MTT) colorimetric assay, as described previously [21]. MTT dissolved in PBS was added to the adherent cells in microtitre wells (20 µl per well) at the end of the experimental period.…”