Elsevier

Neuroscience

Volume 162, Issue 2, 18 August 2009, Pages 462-471
Neuroscience

Sensory System
Research Paper
MKK3, an upstream activator of p38, contributes to formalin phase 2 and late allodynia in mice

https://doi.org/10.1016/j.neuroscience.2009.05.008Get rights and content

Abstract

Spinal p38 mitogen activated (MAP) kinase plays a key role in chronic pain behavior. However, clinical development of p38 inhibitors has been hindered by significant toxicity. To evaluate alternative strategies of p38 regulation, we determined if known upstream activators of p38 (mitogen activated kinase kinase [MKK] 3 and MKK6), are involved in development and maintenance of pain and spinal p38 phosphorylation. Acute pain behaviors were not altered in MKK3 or MKK6 deficient mice. The phase 2 formalin response was delayed in MKK3-/- mice, but unchanged in magnitude, while the response remained normal in MKK6-/- mice. More striking, late formalin allodynia (3–18 days post-injection) was prominent in wild type and MKK6-/- mice, but was delayed for several days in MKK3-/- mice. In wild type, but not MKK3-/- mice, intraplantar formalin elicited increases in ipsilateral spinal MKK3/6 phosphorylation acutely and again at 9 days postinjection. Phosphorylation of MKK3/6 correlated with phase 2 formalin behavior. Wild type (WT) and MKK3-/- mice both expressed increases in spinal phosphorylated p38, however in WT mice this response began several days earlier, and was of higher magnitude and duration than in MKK3-/- mice. This phosphorylation correlated with the late allodynia. Phosphorylated MKK3/6 was detected only in astrocytes, given that phosphorylated p38 (P-p38) is usually not seen in astrocytes this argues for astrocytic release of soluble mediators that affect p38 phosphorylation in microglia. Taking these data together, MKK3, but not MKK6, is necessary for normal development of chronic pain behavior and phosphorylation of spinal p38.

Section snippets

Animals

Mice (20–30 g, male) were gang housed on a 12-h light/dark cycle and fed food and water ad libitum. Knockout (KO) mice for MKK3 and MKK6 were originally generated at Yale University, New Haven, CT, USA (Lu et al 1999, Tanaka et al 2002) where they were backcrossed onto a C57BL/6 line obtained from Charles River Laboratories (Wilmington, MA, USA). MKK3-/- and MKK6-/- mice were bred at UCSD and genotyped according to standard protocols available at the Jackson Laboratories web site: //www.jax.org/imr/supp_proto.html

Thermal and mechanical stimuli

Mean thermal latency and mechanical withdrawal threshold for MKK3-/- and MKK6-/- mice did not differ from that of WT mice (Table 1). Also, no differences were seen between right and left paws (data not shown). Thus, escape responses to acute nociceptive stimulation were unaffected by either MKK3 or MKK6 deficiency.

Formalin response phase 1

The phase 1 response (1–9 min postinjection) is the acute behavioral response to intraplantar formalin and, to a great extent, reflects the degree of primary afferent fiber activity (

Discussion

These studies explored whether the kinases that regulate p38 play contribute to pain behavior in mice. Our major findings are (1) MKK3 and MKK6 do not participate in acute pain; (2) MKK3, but not MKK6, participates in the initiation of formalin phase 2 behavior and the subsequent prolonged allodynia and its phosphorylation correlates with phase 2 activity; (3) lack of P-MKK3 reduces and delays spinal p38 phosphorylation post-injury; and (4) P-MKK3/6 in mice is expressed in astrocytes.

Activation

Acknowledgments

This work was supported by National Institutes of Health grant NS048563 (L.S.S. and G.S.F.) and 5R01AI070555 (G.S.F.). We thank Dr. Camilla Svensson for many valuable discussions concerning the data.

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