Fig 2: Downregulation of CCN2 by reduced dermal fibroblast size is mediated by impaired YAP/TAZ nuclear translocation. (a) YAP/TAZ nuclear translocation is impeded by reduced fibroblast size. Dermal fibroblasts were treated with DMSO (CTRL) or Lat-A (180 nM) for 24 hours. YAP and TAZ protein levels were determined by immunostaining and quantified by ImageJ (National Institutes of Health, Bethesda, MD). YAP and TAZ protein levels were expressed in staining intensity after normalization by cell count. Nuclei showed blue fluorescence (DAPI). Data are representative of three independent experiments. Mean ± SEM. ∗P < 0.05. Bar = 50 μm. (b) Downregulation of CCN2 by reduced dermal fibroblast size is reversed by YAP/TAZ nuclear translocation. Impeded YAP/TAZ nuclear translocation was restored by overexpression of YAP/TAZ-mutant plasmids, which allow for constitutive YAP/TAZ nuclear translocation. CCN2 protein levels were determined by immunostaining and quantified by ImageJ. CCN2 protein levels were expressed in staining intensity after normalization by cell count. Nuclei were stained with DAPI (blue). Data are representative of three independent experiments. Mean ± SEM. ∗P < 0.05. Bar = 50 μm. (c, d) Cells were transfected with control and YAP/TAZ-mutant plasmids for 48 hours. (c) Transfected cells were embedded in intact collagen gel and rhMMP-1‒treated partially broken collagen gel. (d) Transfected cells were plated on regular tissue culture plate (CTRL) or PLL-coated plate. CCN2, YAP, and TAZ protein levels were determined by capillary electrophoresis immunoassay. CCN2 and β-actin levels were determined from cytoplasmic protein fractions, and YAP/TAZ levels were determined from nuclear protein fractions. The chemiluminescence signal of the specific protein from the capillary tube was obtained directly from the capillary system (Compass software, ProteinSimple, Santa Clara, CA), and the data were displayed by lane in a virtual-blot image, which was simulated using the signal intensity (chemiluminescence) of the specific protein. The tables show the quantitative results from the chemiluminescence signals detected by the specific protein from the capillary tubes. The chemiluminescence signal of the CCN2 protein was normalized to β-actin (loading control), and the data were expressed as a percent of control (bar charts). Data are representative of four independent experiments. Mean ± SEM. ∗P < 0.05. 3D, three-dimensional; COL-1, type 1 collagen; CTRL, control; Lat-A, Latrunculin-A; mt, mutant; PLL, poly-L-lysine; rhMMP-1, recombinant human matrix metalloproteinase 1.

Fig 3: Restoration of dermal fibroblast size reversed the inhibition of YAP/TAZ nuclear translocation and reduced CCN2 expression. Dermal fibroblasts were treated with DMSO (CTRL) or Lat-A (180 nM) for 24 hours. After extensive washing, fresh culture media was added for 24 hours. (a) Dermal fibroblast size is restored by removal of Lat-A. (b) Restoring dermal fibroblast size reversed the impediment of YAP/TAZ nuclear translocation. (c) Restoration of dermal fibroblast size reversed CCN2 expression. Cells were immunostained with CCN2 (red) or phalloidin (red), alongside YAP/TAZ (green) and were quantified by ImageJ (National Institutes of Health, Bethesda, MD). Protein levels were expressed in staining intensity after normalization by cell count. Nuclei were stained with DAPI (blue). Data are representative of three independent experiments. Mean ± SEM. ∗P < 0.05. Bar = 50 μm. CTRL, control; Lat-A, Latrunculin-A.

Fig 4: Downregulation of YAP/TAZ, CCN2, and COL-1 in aged human skin in vivo. (a) Human skin biopsies were obtained from young (left, n = 6) and aged (right, n = 6) sun-protected buttock skin. Nanoscale images of collagen fibrils were imaged by AFM (top). Collagen fiber (white), elastin fiber (green), and fibroblast morphology were imaged by multiphoton laser scanning fluorescence microscopy (second from top). YAP/TAZ (middle), CCN2 (second to last), and type I procollagen (bottom) protein expressions were assessed by immunohistochemistry. Images are representative of six subjects from each age group. YAP/TAZ, CCN2, and COL-1 protein levels were quantified by ImageJ (National Institutes of Health, Bethesda, MD) and expressed in the percent of positive staining. Data represent six young and six aged subjects. Mean ± SEM. ∗P < 0.05. Bars for AFM = 200 nm, for multiphoton laser scanning fluorescence microscopy = 25 μm, for YAP/TAZ = 50 μm, for CCN2 = 50 μm, and for COL-1 = 50 μm. (b) YAP/TAZ immunofluorescence staining in young and aged human skin. Blue fluorescence with dot lines delineates the nuclei (DAPI). YAP/TAZ protein levels were quantified by ImageJ and expressed in the percent of positive staining. Note the less YAP/TAZ nuclear staining in the aged dermis. Data represent six young and six aged subjects. Mean ± SEM. ∗P < 0.05. Bar = 25 μm. (c) Age-related reduced fibroblast size concomitantly downregulates YAP/TAZ activity and CCN2 expression in aged human skin. Age-related collagen fragmentation is a prominent feature of aged human skin, which impairs cell‒collagen interaction and alters fibroblast shape and size. Reduced fibroblast size leads to numerous alterations, including impediments of YAP/TAZ activity. Impaired YAP/TAZ activity downregulates CCN2 expression, which in turn contributes to loss of collagen production, a prominent feature of human dermal aging (see Discussion for details). (d) YAP and TAZ mRNA expression in young and aged human skin in vivo. Total RNA was prepared from young and aged sun-protected buttock skin. YAP and TAZ mRNA levels were quantified by real-time RT-PCR and normalized to the housekeeping gene 36B4, as an internal control for quantification (lower left). Data are expressed as mean ± SEM. N = 4. AFM, atomic force microscopy; COL-1, type 1 collagen; FB, fibroblast.