Download Bone Pain Is Mediated by TRPV4 via Aquaporin

Bone Pain Is Mediated by TRPV4 via Aquaporin
1
+1 Wakabayashi H, 1Uchida A, 2Yoneda T
Orthopedic Surgery Mie University Graduate School of Medicine, 2Department of Biochemistry Osaka University Graduate School of Dentistry,
Senior author [email protected]
Introduction
Cancers including breast, prostate and lung cancers frequently
metastasize to bone and cause diverse complications. Among these
complications, bone pain is frequently accompanied with cancer
metastasis to bone and recognized as a major clinical problem as
survival of cancer patients prolongs. Bone pain can be caused by
noxious chemical and/or mechanical stimuli produced by cancer cells
and associated inflammatory cells. Our earlier studies showed that the
transient receptor potential vanilloid 4 (TRPV4), osmosensing receptor,
plays an important role in inducing bone pain associated with cancer cell
colonization in bone. We presented at ORS 2007 that TRPV4-deficient
mice were tolerable in bone pain-related behavior assays.
Phosphorylated-ERK (p-ERK) immunoreactive neurons, markers for
neural activation by noxious stimuli, were significantly decreased in
tumor-bearing side in the primary afferent neurons in TRPV4-deficient
mice compared with wild type mice. Recent studies suggest that
aquaporins (AQP) play a role in causing edema by increasing membrane
water permeability. It is reported that TRPV4 regulates AQP5
abundance under hypotonic condition in epithelia of the lung. In the
present study, we investigated the mechanism of TRPV4 via AQP in the
induction of bone pain associated with cancer colonization in bone.
Materials and Methods
Animal model of cancer-induced bone pain
Lewis lung cancer cells (1X105 cells/10 µl PBS) were inoculated into the
bone marrow cavity of right tibiae in mice under general anesthesia. Left
tibiae received PBS. Tumor growth was monitored weekly by
radiological examinations.
TRPV4 expression and aquaporins expression by RT-PCR
Total RNAs from primary afferent neurons (DRG: dorsal root ganglia),
the tumor-inoculated bone and non-tumor bone were extracted from
C57BL/6 mice 2 weeks after tumor inoculation. Tumor cells total RNAs
were extracted from lewis lung cancer cells. Reverse transcription was
performed. PCR experiments from DRG were done using specific
primers for mouse TRPV4. PCR experiments from cells and bone were
done using specific primers for mouse AQP0 to AQP9.
Aquaporin expression in bone by immunohistochemistry
To examine the expression of AQP in the bone and bone tumor, we
performed immunohistochemical examination in the tumor-inoculated
bone and non-tumor bone.
p-ERK expression in F11cells
We investigated whether hypo or hyper stimuli induced phosphorylated
ERK expression following TRPV4 activation in immunoblot analysis.
To examine this, we used F11 cells, which is a hybridoma of mouse
neuroblastom and rat dorsal root ganglion cells. F11 cells expressed
TRPV4 mRNA but not TRPV1 by RT-PCR. Therefore, the effects of
TRPV1 can be excluded in F11 cells.
Results
Animal model of cancer-induced bone pain
Radiological examinations demonstrated that lewis lung cancer cells
aggressively colonized and destroyed bone (Figure 1).
TRPV4 expression in primary afferent neurons (DRG) by RT-PCR
TRPV4 expression was increased in the tumor-bearing side DRG
compared with the non-tumor-bearing side DRG.
Aquaporin expression in tumor cells and bone by RT-PCR
RT-PCR displayed AQP1, 3 and 8 in lewis lung cancer cells and tumorinoculated bone. And AQP1, 3 and 8 expressions were increased in the
tumor-inoculated bone compared with non-tumor bone (Figure 2).
Aquaporin expression in bone by immunohistochemistry
Tumors growing in bone marrow cavity displayed AQP1 and AQP3
expression but not AQP8 expression. AQP1 and AQP3 expression was
increased in tumors growing in bone marrow cavity compared with bone
marrow without tumor. From these results, it is speculated that increased
AQP1 and/or AQP3 expression in bone that is caused by tumor growth
activates TRPV4.
p-ERK expression in F11cells
When F11 cells were treated with hypotonic stimuli, we observed that pERK expression in F11 cells was increased. And ruthenium red, which is
a non-selective TRPV4 antagonist, decreased p-ERK expression induced
by hypotonic stimuli in a dose-dependent manner (Figure 3). Hypertonic
stimuli, as well as hypotonic stimuli, increased p-ERK expression,
which is inhibited by ruthenium red, in F11 cells.
Discussion and Conclusions
Our results suggest that the increased AQPs expression in tumor
inoculated bone induced hypo- or hypertonic conditions in bone, and
activated TRPV4 and p-ERK expression in the primary afferent neurons.
Figure1 : Two weeks after tumor inoculation in bone.
X-ray showed that tumor cells aggressively
colonized and destroyed bone.
Figure2 : AQP1, 3 and 8 expressions by RT-PCR.
AQP1, 3 and 8 expressions were increased in the tumor-inoculated bone
(Tumor +) compared with non-tumor bone (Tumor -).
AQP1
AQP3
AQP8
GAPDH
Tumor
+
-
Figure3 : Phosphorylated ERK expression in immunoblot analysis.
Hypotonic stimuli increased p-ERK expression in F11 cells. Ruthenium
red (RR), a non-selective TRPV4 antagonist, decreased p-ERK
expression induced by hypotonic stimuli in a dose-dependent manner.
p-ERK
ERK
Control 30%hypo RR1µM RR10µM
Poster No. 1606 • 55th Annual Meeting of the Orthopaedic Research Society