In the skin, sensory neurons are in charge of detecting external stimuli, but they do not go it alone. A new line of research shows that skin cells, and the extracellular matrix proteins they produce, may be important modulators of pain sensation. A study published online July 3 in Nature Neuroscience, from Gary Lewin, Max Delbrück Center for Molecular Medicine, Berlin, Germany, and Jing Hu, Center for Integrative Neuroscience, Tübingen, Germany, reveals that keratinocytes in the epidermis produce a unique extracellular matrix that inhibits the responsiveness and branching of mechanosensitive neurites, including those responsible for pain. The researchers pinpointed the matrix component laminin-332 as the factor responsible for the inhibition.
Besides revealing a new role for the matrix in corralling sensory neuron growth and activity, the study offers potential insight into the painful skin disease epidermolysis bullosa, which is caused by loss of laminin-332 expression.
In the new study, first authors Li-Yang Chiang and Kate Poole, and their coworkers, used whole-cell patch clamp techniques to measure mechanosensitive currents in dorsal root ganglion (DRG) sensory neurons in culture. They found that the laminin matrix produced by mouse keratinocytes, but not that from other kinds of cells, suppressed one type of mechanosensitive current, the rapidly adapting (RA) current. Further studies tracked that inhibition to laminin-332 (formerly called laminin-5), an isoform specific to keratinocyte-derived matrix. In skin, keratinocytes reside in the outer layer, the epidermis, which is innervated by nociceptors. Therefore, these results raise the possibility that laminin-332 dampens the response of epidermal nociceptors, acting as a brake on painful sensations.
Mechanotransduction is mediated via poorly understood ion channels, and there are two views about how the channels are opened. One view holds that stretching the neuronal membrane results in channel opening. However, Lewin told PRF that there is also evidence that mechanosensory channels are linked to the extracellular matrix and open when force is transferred to them from the matrix. Previously, Lewin, Hu, and their colleagues reported that the appearance of mechanoresponsive currents in DRG sensory neurons (and some nociceptors) requires a protein filament that tethers neurons to the matrix (Hu et al., 2010).
The group wondered whether that tether might be missing in the keratinocyte matrix. Indeed it was: Transmission electron microscopy (TEM) of sensory neurons cultured on laminin showed that when the matrix contained laminin-332, the tethers disappeared.
To figure out how laminin-332 might be having its inhibitory effect, the researchers plated sensory neurons on a surface micro-printed with a laminin grid. Strikingly, RA mechanosensitive currents could be evoked from neurites growing on laminin-332–free stripes, but neurites sitting on neighboring laminin-332 stripes rarely showed those currents. That was true even for neurites in the same cell. Because stripes were only 25 μm apart—a tiny space compared to the area covered by a neuron’s receptive field—the authors conclude that laminin-332 acts on neurites locally, presumably disrupting attachment of the tether to the matrix and thus preventing ion channels from opening.
In addition, neurites grew preferentially on stripes lacking laminin-332. Growth cones regularly branched on the permissive matrix, but rarely sent new neurites into matrix containing laminin-332.
Lewin wonders whether neurites pick their paths by actually “feeling” the shape of the laminin matrix, which atomic force microscopy (AFM) showed became bumpier upon addition of laminin-332. “It could be that the growth cone is sensing mechanical force, like your finger does if you run it over sandpaper,” he said.
In addition to their studies on cultured mouse cells, the team investigated tissue from patients with Herlitz-type junctional epidermolysis bullosa (EB), a skin disease caused by mutations in the genes for any of laminin-332’s three subunits. The lack of laminin-332 leads to extreme skin blistering and pain, and this type of EB is often lethal in the first year of life. The researchers found that laminin matrix from keratinocytes of healthy people dampened RA mechanosensitive currents in cultured neurons, whereas matrix from patients with EB failed to suppress those currents. Further, skin biopsies showed changes in nerve branching: In healthy skin, laminin-332 was present at the junction between dermis and epidermis, and sensory fibers that grew across that border were usually unbranched. In patients with EB, fibers branched at the border.
The observed increases in neuron excitability and branching may help to explain the pain that marks EB. The findings may also shed light on how sensory neurons are regulated normally. Previously, Lewin says, laminin-332 was seen strictly as a structural protein that helps to maintain skin integrity; now, his group’s findings reveal that it also holds sway over sensory neurons. For neurons that lie just below the skin’s surface, he speculates, laminin-332 might create “an endogenous inhibition mechanism…to make sure those fibers are not oversensitive.”
This study joins a number of other new findings on the role of keratinocytes in modulating sensory neurons and pain. Recently, researchers from the labs of Phillip Albrecht and Frank Rice at Albany Medical College and Integrated Tissue Dynamics, Rensselaer, New York, reported that calcitonin gene-related peptide β (CGRPβ) from keratinocytes—previously thought to be CGRPα from neurons—may drive a variety of chronic pain conditions (Hou et al., 2011).
Finally, Lewin’s study hints that the extracellular matrix may constitute a new target for treating peripheral pain. In the case of laminin-332, Lewin says that small molecules mimicking its action, applied topically, could possibly serve as analgesic agents for a wide range of pain conditions. He notes that, in addition to EB, other diseases such as diabetic neuropathy involve changes in epidermal innervation.
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Chih-Cheng Chen, Academia Sinica
This comment was coauthored
This comment was coauthored with Phillip LeDuc, Department of Mechanical and Biomedical Engineering, Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania.
Chiang et al. exhibited a series of elegant experiments to demonstrate that the surface-bound laminin-332 has a critical role in suppressing rapidly adapting mechanosensitive current on sensory afferents. The authors found that a laminin-332-containing substrate disrupted the mechanotransduction tether that is required for rapidly adaptive mechanosensitive current. In contrast, pretreatment of neurons with soluble laminin-332 failed to inhibit the mechanosensitive current. This effect of laminin-332 is believed to be coupled to the activation of a3b1 and a6b4 integrin receptors. Paradoxically, when neurons were cultured on a laminin/laminin-332 mixture substrate, blocking the G domain of laminin-332 with CM6 antibodies, this did not prevent its suppression of the rapidly adaptive mechanosensitive current.
The molecular machinery responsible for mechanotransduction in mammalian sensory neurons is still largely unknown. Accumulated data have supported one hypothesized model that stretch-activated ion channels are tethered to both extracellular matrix and intracellular architecture (Khalsa et al., 2004; Lin et al., 2009; Hu et al., 2010). The beauty of the work by Chiang et al is they identified a specific matrix protein laminin-332 in coordinating mechanotransduction and branching of sensory terminals in the skin. Furthermore, this protein might be related to the extreme pain experienced by patients with Herlitz-type junctional epidermolysis bullosa. In this area, laminin-332 and activation of the integrin receptors might be useful for pain control in these patients. Also, an incentive for analgesic drug development in this area is that the drug could be simply applied on the skin via cream or patch, as the target site is on the epidermis. Also, a more general application of laminin-332 might be extended to treat mechanical hyperalgesia and allodynia in a wide range of chronic pain patients.
This set of studies brings up some very intriguing questions. These involve the direct suppression on mechanosensitive current by laminin-332 and whether that comes from the soluble laminin-332. This did not appear to show inhibitory effect on mechanosensitive current and also CM6 antibodies did not have an effect on the suppression of rapidly adaptive mechanosensitive current for sensory neurons cultured on a laminin-332-containing substrate.
One interesting point that could lead to further studies is the effect of substrate stiffness, as this might be very important. In our recent work, we have found that substrate stiffness plays an important role on neurite outgrowth in cultured sensory neurons of dorsal root ganglia. In the study of Chiang et al., laminin-332 markedly altered the network structure of the laminin matrix and thus might alter the matrix stiffness in microenvironment as well. Therefore, sensory neurons might be affected by different substrate stiffnesses for laminin substrate when cultured on laminin-332-containing substrates or co-cultured with keratinocytes. Although substrate stiffness is not often considered in many neurobiology studies, it does play an important role in cell-matrix interaction in mechanobiology involving a wide range of cells from bone to heart to stem cells.
One idea that could be a very interesting future study is to investigate the inhibitory effect of laminin-332 with respect to substrate stiffness. This could be accomplished through pursuing similar studies with elastomeric substrates. Single neurites of neurons grown on an elastomeric substrate could be stretched locally and recorded for the mechanotransduction via whole-cell patch-clamp techniques. We have successfully used fibronectin-coated PDMS as an elastomeric substrate for this purpose. Under this elastomeric condition, the stretch-activated mechanotransduction can be only seen in 7-35 percent of neurons, depending on the culture status (Lin et al., 2009). If researchers coated laminin-332 on different stiffness PDMS substrates, they would be able to probe the specific effect of the extracellular matrix proteins on stretch-activated mechanotransduction via electrophysiology.
In summary, the work by Chiang et al. has significantly advanced our understanding in neurosensory mechanotransduction. To date, studies in this field are still classifying and characterizing different types of mechanosensitive currents and are often descriptive. The finding of laminin-332 in suppression of mechanosensitive current is a breakthrough, as this will facilitate more insightful research into the heterogeneous roles of the extracellular matrix proteins on mechanotransduction. In addition, this protein is required for maintaining the structural integrity of the skin, and might modulate the sensory afferent patterning in dermis and epidermis domains as well as affect mechanical nociception.
References:
Karen Lines, GoToMassage.com
I have a Fibromyalgia client
I have a fibromyalgia client who had childhood rheumatic fever. Her heart valves were not affected, her knees are severely affected, she is anorexic and suffers migraines frequently. Is it possible post rheumatic fever pain could result from endotoxin reaction and laminin destruction in the subcutaneous skin? I offer the paper below.
Cytotoxic mAb from rheumatic carditis recognizes heart valves and laminin.
Galvin JE, Hemric ME, Ward K, Cunningham MW.
J Clin Invest. 2000Jul;106(2):217-24.
Thank you for your time considering this question.
Uhtaek Oh, Seoul National University
Lewin’s laboratory has been
Lewin’s laboratory has been studying mechanisms underlying mechanosensation for a long time. This time he and his colleagues have undertaken very interesting experiments that no other laboratories would think of. First, his colleagues sought to identify the interaction between mechanoreceptors or nociceptors and extracellular matrix such as laminins. Knowing the fact that keratinocytes inhibit mechanosensitive (MS) currents in DRG neurons, the Lewin’s group pin-pointed laminin-332 among various laminins secreted by keratinocytes that specifically inhibit MS currents in DRG neurons. Then, they adopted the grid analysis where they printed different laminins etched in different lines. On the grid, DRG neurons were cultured and tested for MS currents when each grid was touched with a mechanical stimulation probe. Convincingly enough, when laminin-332 grid was touched, MS currents were depressed whereas MS currents were present when laminin grid without laminin-332 was touched. Technically, this is type of experiment is very difficult because printing laminin strips on the glass in small scale requires state-of-the-art precision.
Another important issue of the article is the clinical implication of this finding. The results of the present article may explain why severe pain ensues when laminin-332 is mutated. The authors explain that when laminin-332 is mutated, then its suppression of MS currents in nociceptors is lifted, which evokes hyperalgesia to mechanical stimuli.
In this paper, Lewin’s group again introduces state-of art techniques to prove the specific functional interaction between extracellular matrix and sensory nerves in mediating mechanosensation.