Highly homologous Mycobacterium tuberculosis chaperonin 60 proteins with differential CD14 dependencies stimulate cytokine production by human monocytes through cooperative activation of p38 and ERK1/2 mitogen-activated protein kinases.

Tuberculosis is a chronic inflammatory and destructive disease caused by infection with Mycobacterium tuberculosis. We have previously shown that the mycobacterial chaperonin (Cpn)60.1 and 60.2 proteins stimulate human monocytes to secrete pro-inflammatory cytokines. Identification of the cellular mechanisms that contribute to the chronic inflammation characterised by myobacterial infection is therefore of potential therapeutic benefit. In the present study we have investigated the role of the extracellular signal-regulated (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) families in Cpn60-induced cytokine synthesis, and have compared the effects of the bacterial proteins with those of lipopolysaccharide (LPS). Exposure to Cpn60.1, Cpn60.2 or LPS enhanced ERK1/2 activation with increases in phosphorylation evident between 10 and 30 min and maximal after 60-90 min stimulation. Phosphorylation of ERK1/2 in Cpn60-stimulated monocytes was maintained whereas ERK1/2 was rapidly dephosphorylated in LPS-stimulated cells. Exposure to the chaperonins also caused rapid activation of p38(mapk) with kinetics of phosphorylation comparable to those observed in response to LPS. Selective inhibitors of p38(mapk) (SB203580) or of MEK1/2, the direct upstream activator of ERK1/2 (PD98059), reduced the synthesis of IL-1beta, TNFalpha, IL-6 and IL-8 induced by either the chaperonins or LPS. Experiments in which cells were exposed to a combination of both inhibitors led to a nearly complete abrogation of agonist-induced cytokine synthesis. These results show that the p38(mapk) and ERK1/2 signalling pathways are important regulators of the cellular response to mycobacterial chaperonins and that these pathways cooperate to regulate pro-inflammatory cytokine production by human monocytes.

A specific mechanomodulatory role for p38 MAPK in embryonic joint articular surface cell MEK-ERK pathway regulation.

Mechanisms regulating cell behavior and extracellular matrix composition in response to mechanical stimuli remain unresolved. Our previous studies have established that the MEK-ERK cascade plays a specific role in the mechano-dependent joint formation process by promoting the assembly of pericellular matrices reliant upon hyaluronan (HA) for their integrity. Here we demonstrate: (i) novel cross-talk between p38 MAPK and MEK-ERK signaling pathways that is specific for mechanical stimuli and (ii) a role for p38 MAPK in facilitating HA production by cells derived from the articular surface of embryonic chick tibiotarsal joints. We find that p38 MAPK blockade restricts pericellular assembly of HA-rich matrices and reduces basal as well as mechanical strain-induced release of HA. p38 MAPK blockers potentiated early strain-induced increases but restricted sustained increases in MEK/ERK phosphorylation at later times; c-Fos hyperphosphorylation at threonine 325 was found to parallel this p38 MAPK-mediated modulation of ERK activation. In contrast, p38 MAPK inhibitors had no detectable effect on the ERK activation induced by fibroblast growth factor 2 or pervanadate, a phosphatase inhibitor, and MEK inhibitors did not influence p38 MAPK phosphorylation, confirming both the specificity and unidirectionality of p38 MAPK-ERK cross-talk. Immunochemical and immunoblotting studies revealed constitutive p38 MAPK activation in cells at, or derived from, developing articular joint surfaces. Unlike the MEK-ERK pathway, however, p38 MAPK was not further stimulated by mechanical stimulation in vitro. Thus, p38 MAPK specifically facilitates ERK activation and downstream signaling in response to mechanical stimuli. These results suggest that constitutively active p38 MAPK serves an essential, permissive role in mechanically induced changes in ERK activation and in the accumulation of HA-rich extracellular matrices that serve a key role in joint development.

Selective activation of the MEK-ERK pathway is regulated by mechanical stimuli in forming joints and promotes pericellular matrix formation.

It is well established that local modification of extracellular matrix (ECM) hyaluronan composition is vital in the regulation of cell behavior. Indeed, the formation of articulating chick joint cavities, which requires mechanical stimuli derived from skeletal movement, is dependent upon the accumulation of an ECM rich in hyaluronan (HA). However, the mechanisms responsible for such precise mechano-dependent regulation of cell behavior and the formation of a HA-rich ECM remain undefined. Here we show that extracellular-regulated kinase 1/2 (ERK1/2) is selectively activated in cells at sites of cavity formation and activity diminished by in ovo immobilization that induces cartilaginous fusion across presumptive joint interzones. In vitro analyses offer mechanistic support for the role of mechanical stimuli in promoting a MEK-dependent activation of ERK1/2. In addition, our direct regulation of ERK1/2 phosphorylation status via modulation of its up-stream "classical cascade" activator either pharmacologically or by transfection with dominant negative or constitutively active Mek confirms the essential role for ERK1/2 activation in the elaboration of HA-rich pericellular matrices. Together, our findings demonstrate that the MEK-ERK pathway, regulated by mechanical stimuli, controls HA-rich matrix assembly. The precision of ERK1/2 activation selectively distinguishing cells at the joint line suggests that it directly contributes to the loss of tissue cohesion essential for generating HA-rich cavities between joint elements during their development.

Diverse range of fixed positional deformities and bone growth restraint provoked by flaccid paralysis in embryonic chicks.

Pancuronium bromide (PB) is used in neonates and pregnant women to induce limp, flaccid paralysis in order to allow mechanical ventilation during intensive care. Such non-depolarizing neuromuscular blocking drugs are administered to 0.1% of all human births in the UK. In this study, we examined PB effects on skeletal development in chick embryos. PB treatment produced skeletal deformities associated with significant reduction in longitudinal growth of all appendicular elements. This was associated with greater cartilage to bone ratios, indicating a preferential reduction in osteogenesis. PB also increased the incidence of knee joint flexion and tibiotarsal joint hyperextension. In addition to limb, spinal and craniofacial deformities, flaccid immobility appears to convert the normal geometric pattern of weight gain to a simple arithmetic accretion. This novel study highlights the potentially harmful effects of pharmacologically induced flaccid immobility on chick embryonic skeletal development. Whilst in ovo avian development clearly differs from human, our findings may have implications for the fetus, premature and term neonate receiving such non-depolarizing neuromuscular blocking drugs.