Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)-beta. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Galpha(12/13)) in cardiomyocytes suppressed pressure overload-induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF-beta, connective tissue growth factor, and periostin) and Ang-converting enzyme (ACE) was suppressed by the functional inhibition of Galpha(12/13). The expression of these fibrogenic genes through Galpha(12/13) by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G-protein-coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Galpha(12/13) in cardiomyocytes by the extracellular nucleotides-stimulated P2Y(6) receptor triggers fibrosis in pressure overload-induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF-beta.

Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response 1,2 . Rab5 is known as a key regulator of the early endocytic pathway 3 and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment 4 ; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominantnegative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.To make Rab5 activity visible in living cells, we developed a genetically encoded fluorescence resonance energy transfer (FRET) probe, designated Raichu-Rab5. The probe comprised a modified yellow fluorescent protein (YFP) called Venus, the amino-terminal Rab5-binding domain of EEA1, a modified cyan fluorescent protein (CFP) called SECFP, and Rab5 (Fig. 1a). In this probe design, an increase in Rab5-GTP results in an increase in FRET, which can be represented by the 525 nm/475 nm emission ratio. Characterization of Raichu-Rab5 was conducted similarly to that of other Raichu probes reported previously 5,6 . In comparison with the wild-type Rab5 probe, Raichu-Rab5-Q79L-which lacks GTPase activity-had an increased FRET efficiency, whereas Raichu-Rab5-S34N-which shows a reduced affinity for guanine nucleotides-had a decreased FRET efficiency, as expected ( Supplementary Fig. 1a). The GTP loading of the Rab5 probes correlated well with that of the authentic Rab5 proteins ( Supplementary Fig. 1b), and the GTP loadings obtained here were similar to those reported previously 7 . Next, we examined the sensitivity of Raichu-Rab5 to guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) (Supplementary Fig. 1c). Rab5 GEFs such as Rabex-5, Rin1 and Gapex-5 induced a dose-dependent increase in the FRET efficiency of Raichu-Rab5. In contrast, the expression of Rab5 GAPs such as RabGAP-5 and RN-tre decreased the FRET efficiency in a dose-dependent manner. These results indicate that Raichu-Rab5 is capable of monitoring the balance between GEF and GA...

The efficient engulfment of apoptotic cells by professional or nonprofessional phagocytes is critical to maintain mammalian homeostasis. To identify molecules involved in the engulfment of apoptotic cells, we established a retrovirus-based expression cloning system coupled with the engulfment assay. By screening a cDNA library of a mouse macrophage cell line, we identified two small GTPase family members (RhoG and Rab5) that enhanced the engulfment of apoptotic cells. By examining other small GTPase family members, we found that Rac1 enhanced the engulfment of apoptotic cells, whereas RhoA inhibited the process. Accordingly, the expression of a dominant-negative form of RhoG or Rac1 in primary macrophage cultures severely reduced the ability of the macrophages to engulf apoptotic cells, and a dominant-negative form of RhoA enhanced the process. These results indicated that the efficient engulfment of apoptotic cells requires the concerted action of small GTPase family members. We demonstrated previously that NIH3T3 cells expressing the ␣ v ␤ 3 integrin efficiently engulf apoptotic cells in the presence of milk fat globule epidermal growth factor 8 via a phosphatidylserine-dependent mechanism. The dominant-negative form of RhoG or Rac1 inhibited this process, which suggested RhoG and Rac1 are also involved in the integrin-mediated engulfment.Apoptosis is a cell-autonomous process that eliminates harmful or useless cells in metazoans (1, 2). It contributes to development, tissue remodeling, and the resolution of inflammation. The apoptotic program is triggered by a variety of stimuli, including anti-cancer drugs and death factors, and is mediated by a family of caspases (3, 4). Caspases cleave a set of cellular proteins such as cytoskeletal and structural proteins, which results in the morphological changes that characterize apoptotic cell death (5). The activation of caspases is also responsible for the extensive degradation of chromosomal DNA, another hallmark of apoptosis, which is mediated by a specific DNase (caspase-activated DNase, CAD) 3 (6). Apoptotic cells are rapidly engulfed by professional phagocytes (macrophages and immature dendritic cells) or less efficiently by nonprofessional phagocytes such as fibroblasts and epithelial cells (7,8). This prompt engulfment seems to prevent the release of potentially noxious or immunogenic intracellular contents from dying cells, thereby preserving the integrity and function of the surrounding tissues. Phagocytes engulf apoptotic cells but not healthy cells, indicating that the apoptotic cells display an "eat me" signal(s) on the cell surface, and the phagocytes directly or indirectly recognize this signal. Among the variety of molecules that have been proposed as an eat me signal, phosphatidylserine (PS) is the best candidate (9). In living cells, PS is confined to the inner leaflet of the plasma membrane, but it is quickly exposed to the cell surface when cells undergo apoptosis. We showed previously that a factor called milk fat globule epidermal growth factor ...

digested by collagenase/trypsin, and the digested cardiac cells were allowed to attach to the plates overnight. The attached cells included macrophages and myofibroblasts (positive for α smooth muscle actin [αSMA]) as well as other cardiac cells (Supplemental Figure 1B). Notably, cardiac myofibroblasts seemed to be more difficult than cardiac macrophages to collect using our isolation method from infarcted hearts because, as revealed by our immunohistochemical analysis, the number of cardiac myofibroblasts was the same as that of cardiac macrophages in the infarcted area (Supplemental Figure 1C). When the overnight-attached cells were cultured in 10% FBS/DMEM for more than 6 days, almost all of the cells on the plates were positive for αSMA and SM22α, 2 myofibroblast marker proteins (18, 19) (>97.9% and >93.8%, respectively) (Supplemental Figure 1, D and E), indicating that the cells were primarily composed of cardiac myofibroblasts. This is probably because only myofibroblasts were able to grow rapidly in the culture medium.Isolated cardiac macrophages and myofibroblasts were allowed to engulf fluorescently labeled apoptotic cells, and we assessed the fluorescence taken up by cardiac macrophages and administration promoted the restoration of cardiac function and morphology after MI, suggesting that MFG-E8 is a new therapeutic target for the treatment of MI.

The engulfment of apoptotic cells requires phagocytes to coordinately activate Rho family GTPases that regulate actin dynamics. Here, we used a FRET biosensor to visualize the spatiotemporal activation of Rac1 during engulfment of apoptotic cells. We report that apoptotic cells were usually engulfed by the phagocytes' lamellipodia, where Rac1 was activated. Often, apoptotic cells were engulfed successively at the same lamellipodial site, suggesting the presence of portals for apoptotic cells. At this location, the activated Rac1 was recruited to form phagocytic cups that were comprised of actin patches. When the phagocytic cup was closed, Rac1 was down-regulated, and the actin patches were abruptly broken down. The constitutively active Rac1 remained at phagocytic cup for a longer period than the wild-type Rac1, and the closure of the phagocytic cup was significantly delayed in cells expressing a constitutive active form of Rac1, resulting in inefficient engulfment. These results indicate that activated Rac1 is necessary to assemble F-actin, but closing the phagocytic cup requires Rac1 to be deactivated. apoptosis ͉ FRET ͉ small GTPase