Functional characterization of TRPV4 as an osmotically sensitive ion channel in porcine articular chondrocytes.
OBJECTIVE
Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-permeable channel that can be gated by tonicity (osmolarity) and mechanical stimuli. Chondrocytes, the cells in cartilage, respond to their osmotic and mechanical environments; however, the molecular basis of this signal transduction is not fully understood. This study was undertaken to demonstrate the presence and functionality of TRPV4 in chondrocytes.


METHODS
TRPV4 protein expression was measured by immunolabeling and Western blotting. In response to TRPV4 agonist/antagonists, osmotic stress, and interleukin-1 (IL-1), changes in Ca(2+) signaling, cell volume, and prostaglandin E(2) (PGE(2)) production were measured in porcine chondrocytes using fluorescence microscopy, light microscopy, or immunoassay, respectively.


RESULTS
TRPV4 was expressed abundantly at the RNA and protein levels. Exposure to 4alpha-phorbol 12,13-didecanoate (4alphaPDD), a TRPV4 activator, caused Ca(2+) signaling in chondrocytes, which was blocked by the selective TRPV4 antagonist, GSK205. Blocking TRPV4 diminished the chondrocytes' response to hypo-osmotic stress, reducing the fraction of Ca(2+) responsive cells, the regulatory volume decrease, and PGE(2) production. Ca(2+) signaling was inhibited by removal of extracellular Ca(2+) or depletion of intracellular stores. Specific activation of TRPV4 restored the defective regulatory volume decrease caused by IL-1. Chemical disruption of the primary cilium eliminated Ca(2+) signaling in response to either 4alphaPDD or hypo-osmotic stress.


CONCLUSIONS
Our findings indicate that TRPV4 is present in articular chondrocytes, and chondrocyte response to hypo-osmotic stress is mediated by this channel, which involves both an extracellular Ca(2+) and intracellular Ca(2+) release. TRPV4 may also be involved in modulating the production or influence of proinflammatory molecules in response to osmotic stress.
Duke University Medical Center, Durham, North Carolina 27710, USA.
Mimi N
MN
Phan
Holly A
HA
Leddy
Bartholomew J
BJ
Votta
Sanjay
S
Kumar
Dana S
DS
Levy
David B
DB
Lipshutz
Suk Hee
SH
Lee
Wolfgang
W
Liedtke
Farshid
F
Guilak
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D048429:Cell Size; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology; D015232:Dinoprostone / Q000378:metabolism; D007375:Interleukin-1 / Q000378:metabolism; D023421:Models, Animal; D009995:Osmosis / Q000502:physiology*; D010703:Phorbol Esters / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D013552:Swine; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*
D007375:Interleukin-1; D010703:Phorbol Esters; D050916:TRPV Cation Channels; C008876:phorbol-12,13-didecanoate; D015232:Dinoprostone; D002118:Calcium