The caveolin-1 scaffolding domain peptide decreases phosphatidylglycerol levels and inhibits calcium-induced differentiation in mouse keratinocytes

Abstract

Phospholipase D2 (PLD2) has been found localized in low-density caveolin-rich membrane microdomains. Our previous study suggested that PLD2 and aquaporin 3 (AQP3) interact in these domains to inhibit keratinocyte proliferation and promote differentiation by cooperating to produce phosphatidylglycerol. To examine the effect of membrane microdomain localization on the PLD2/AQP3 signaling module and keratinocyte proliferation and differentiation, we treated mouse keratinocytes with 3 µM cell-permeable caveolin-1 scaffolding domain peptide or a negative control peptide and stimulated cell differentiation using a moderately elevated extracellular calcium concentration (125 uM) to maximally promote differentiation and phosphatidylglycerol production. Cell proliferation, differentiation, total PLD activity, phosphatidylglycerol levels, and AQP3 activity were monitored. The caveolin-1 scaffolding domain peptide itself had no effect on phosphatidylglycerol levels or keratinocyte proliferation or differentiation but prevented the changes induced by a moderately elevated calcium concentration, whereas a negative control did not. The caveolin-1 scaffolding domain peptide had little effect on total PLD activity or glycerol uptake (AQP3 activity). We conclude that the caveolin-1 scaffolding domain peptide disrupts the functional association between AQP3 and PLD2 and prevents both the inhibited proliferation and the stimulated differentiation in response to elevated extracellular calcium levels. The interaction of caveolin-1 and PLD2 is indirect (i.e., lipid mediated); together with the proliferation-promoting effects of caveolin-1 knockout on epidermal keratinocytes, we propose that the caveolin-1 scaffolding domain pepetide exerts a dominant-negative effect on caveolin-1 to alter lipid rafts in these cells.

Figure 1. The caveolin-1 scaffolding domain peptide prevents the calcium-induced inhibition of DNA synthesis.

Keratinocytes were treated for 24 hours with medium containing vehicle (0.1% DMS0) or 3 µM caveolin-1 scaffolding domain peptide (CSDP) or the negative control (Neg) in medium containing 25 µM calcium (Con) or 125 µM calcium (Ca²⁺), as indicated. [³H]Thymidine incorporation into DNA was measured as described in the Materials and Methods. Values are expressed as the percent control and represent the means ± SEM of 12 separate experiments performed in duplicate; *p<0.05, **p<0.01 versus the control value; ++p<0.01 versus calcium alone.

Figure 2. The caveolin-1 scaffolding domain peptide prevents the calcium-induced stimulation of transglutaminase activity.

Keratinocytes were treated for 24 hours with SFKM containing vehicle (0.1% DMSO) or 3 µM caveolin-1 scaffolding domain peptide (CSDP) or the negative control (Neg) in medium containing 25 µM calcium (Con) or 125 µM calcium (Ca²⁺), as indicated. Transglutaminase activity was then measured as described in Materials and Methods. Values are expressed as the percent control and represent the means ± SEM of 6 separate experiments performed in duplicate; *p<0.05 versus the control value.

Figure 3. Caveolin-1 scaffolding domain peptide pretreatment has no effect on calcium-induced IP₃ production.

Keratinocytes were pretreated for 24 hours with SFKM containing vehicle (0.1% DMS0) or 3 µM caveolin-1 scaffolding domain peptide (CSDP). The cells were then treated for 10 minutes with control (25 µM calcium-containing) medium or 1 mM calcium-containing medium (to trigger immediate and maximal calcium-sensing receptor activation), and inositol 1,4,5-trisphosphate levels were measured with a radioreceptor assay as described in Materials and Methods. Values are expressed as the percent control and represent the means ± SEM of 4 separate experiments performed in duplicate; ***p<0.001 versus the control value.

Figure 4. The caveolin-1 scaffolding domain peptide has a minimal effect on calcium-induced PLD activation.

Keratinocytes were preincubated for 24 hours with SFKM containing [³H]oleate and vehicle (0.1% DMS0), 3 μM caveolin-1 scaffolding domain peptide (CSDP) or the negative control (Neg) in medium containing 25 µM calcium (Con) or 125 µM calcium (Ca²⁺) as indicated. [³H]Phosphatidylethanol levels were then measured as described in Materials and Methods. Values are expressed as the percent control and represent the means ± SEM of 5 separate experiments performed in duplicate; **p<0.01 versus the control value.

Figure 5. The caveolin-1 scaffolding domain peptide decreases calcium-increased phosphatidylglycerol levels.

Keratinocytes were treated for 24 hours with SFKM containing vehicle (0.1% DMSO) or 3 μM caveolin-1 scaffolding domain peptide (CSDP) or the negative control (Neg) in medium containing 25 μM calcium (Con) or 125 μM calcium (Ca²⁺) as indicated.. [¹⁴C]Phosphatidylglycerol levels were then measured as described in Materials and Methods. Values are expressed as the percent control and represent the means ± SEM of 3 separate experiments performed in duplicate; **p<0.01 versus the control value; ††p<0.01 versus calcium alone.

Figure 6. The caveolin-1 scaffolding domain peptide has a minimal effect on calcium-induced glycerol uptake.

Keratinocytes were treated for 24 hours with SFKM containing vehicle (0.1% DMS0) or 3 µM caveolin-1 scaffolding domain peptide (CSDP) or the negative control (Neg) in medium containing 25 µM calcium (Con) or 125 µM calcium (Ca²⁺) as indicated. [³H]Glycerol uptake was then measured as described in Materials and Methods. Values are expressed as the percent control and represent the means ± SEM of 4 separate experiments performed in duplicate; **p<0.01 versus the control value.

Figure 7. The caveolin-1 scaffolding domain peptide decreases phosphatidylglycerol levels in calcium-pretreated keratinocytes.

Keratinocytes were pretreated for 24 hours with SFKM containing 125 µM calcium prior to treatment for the indicated times with vehicle (DMSO) or 3µM caveolin-1 scaffolding domain peptide (CSDP) or the negative control (Neg) as indicated. [¹⁴C]Phosphatidylglycerol levels were then measured as described in the Materials and Methods. Values are expressed as the percent vehicle and represent the means ± SEM of 3 separate experiments performed in duplicate; **p<0.01 versus the control or CSDP; †††p<0.001 versus the control or negative control.