Analysis of CCN Functions in Liver Regeneration After Partial Hepatectomy.

The remarkable regenerative capability of the liver has long been appreciated. Upon significant loss of liver tissue, the remnant liver can grow rapidly to restore the original liver mass through a combination of hepatocyte proliferation and hypertrophy to maintain homeostasis. Experimentally, 2/3 partial hepatectomy in mice has been used extensively as a model to dissect the molecular mechanism of liver regeneration and the genetic networks involved. Herein, we describe the protocols for partial hepatectomy and analyses of pertinent CCN protein functions.

Cellular communication network factor 1-stimulated liver macrophage efferocytosis drives hepatic stellate cell activation and liver fibrosis.

Following inflammatory injury in the liver, neutrophils quickly infiltrate the injured tissue to defend against microbes and initiate the repair process; these neutrophils are short lived and rapidly undergo apoptosis. Hepatic stellate cells (HSCs) are the principal precursor cells that transdifferentiate into myofibroblast-like cells, which produce a large amount of extracellular matrix that promotes repair but can also lead to fibrosis if the injury becomes chronic. The matricellular protein cellular communication network factor 1 (CCN1) acts as a bridging molecule by binding phosphatidylserine in apoptotic cells and integrin αv ß3 in phagocytes, thereby triggering efferocytosis or phagocytic clearance of the apoptotic cells. Here, we show that CCN1 induces liver macrophage efferocytosis of apoptotic neutrophils in carbon tetrachloride (CCl4 )-induced liver injury, leading to the production of activated transforming growth factor (TGF)-ß1, which in turn induces HSC transdifferentiation into myofibroblast-like cells that promote fibrosis development. Consequently, knock-in mice expressing a single amino acid substitution in CCN1 rendering it unable to bind αv ß3 or induce efferocytosis are impaired in neutrophil clearance, production of activated TGF-ß1, and HSC transdifferentiation, resulting in greatly diminished liver fibrosis following exposure to CCl4 . Conclusion: These results reveal the crucial role of CCN1 in stimulating liver macrophage clearance of apoptotic neutrophils, a process that drives HSC transdifferentiation into myofibroblastic cells and underlies fibrogenesis in chronic liver injury.

Senescent hepatic stellate cells promote liver regeneration through IL-6 and ligands of CXCR2.

Senescent cells have long been associated with deleterious effects in aging-related pathologies, although recent studies have uncovered their beneficial roles in certain contexts, such as wound healing. We have found that hepatic stellate cells (HSCs) underwent senescence within 2 days after 2/3 partial hepatectomy (PHx) in young (2-3 months old) mice, and the elimination of these senescent cells by using the senolytic drug ABT263 or by using a genetic mouse model impaired liver regeneration. Senescent HSCs secrete IL-6 and CXCR2 ligands as part of the senescence-associated secretory phenotype, which induces multiple signaling pathways to stimulate liver regeneration. IL-6 activates STAT3, induces Yes-associated protein (YAP) activation through SRC family kinases, and synergizes with CXCL2 to activate ERK1/2 to stimulate hepatocyte proliferation. The administration of either IL-6 or CXCL2 partially restored liver regeneration in mice with senescent cell elimination, and the combination of both fully restored liver weight recovery. Furthermore, the matricellular protein central communication network factor 1 (CCN1, previously called CYR61) was rapidly elevated in response to PHx and induced HSC senescence. Knockin mice expressing a mutant CCN1 unable to bind integrin α6ß1 were deficient in senescent cells and liver regeneration after PHx. Thus, HSC senescence, largely induced by CCN1, is a programmed response to PHx and plays a critical role in liver regeneration through signaling pathways activated by IL-6 and ligands of CXCR2.

Quantitative proteomics reveals a Gα/MAPK signaling hub that controls pheromone-induced cellular polarization in yeast.

The mating-specific yeast Gα controls pheromone signaling by sequestering Gßγ and by regulating the Fus3 MAP kinase. Disrupting Gα-Fus3 interaction leads to severe defects in chemotropism. Because Gα concentrates at the chemotropic growth site where Fus3 is required for the phosphorylation of two known targets, we screened for additional proteins whose phosphorylation depends on pheromone stimulation and Gα-Fus3 interaction. Using a mutant form of Gα severely defective in Fus3-binding, GαDSD, and quantitative mass spectrometry, fourteen proteins were identified as potential targets of Gα-recruited Fus3, ten of which were previously implicated in cell polarity and morphogenesis. To explore the biological relevance of these findings, we focused on the Spa2 polarisome protein, which was hypophosphorylated on multiple serine residues in pheromone-treated GαDSD cells. Six sites were mutagenized to create the Spa26XSA mutant protein. Spa26XSA exhibited increased affinity for Fus3, consistent with a kinase-substrate interaction, and Spa26XSA cells exhibited dramatic defects in gradient sensing and zygote formation. These results suggest that Gα promotes the phosphorylation of Spa2 by Fus3 at the cortex of pheromone-stimulated cells, and that this mechanism plays a role in chemotropism. How the Gα-Fus3 signaling hub affects the other putative targets identified here has yet to be determined. SIGNIFICANCE: Previously, interaction between the G alpha protein, Gpa1, and the MAPK of the pheromone response pathway, Fus3, was shown to be important for efficient sensing of the pheromone gradient and for the maintenance of cell polarity during mating. Here we show that the underlying molecular mechanisms involve the phosphorylation of specific cortical targets of Gpa1/Fus3. These have been identified by quantitative phosphoproteomics using a mutant of Gpa1, which is defective in interacting with Fus3. One of these targets is the polarisome protein Spa2. Alanine substitution of the Spa2 phosphorylation sites targeted by Gpa1/Fus3 lead to a dramatic defect in pheromone gradient sensing and zygote formation. These results reveal how the G alpha protein and the MAPK control cell polarity in a prototypical model system. Our results have wider significance as similar mechanisms exist in higher eukaryotes and are involved in important biological such as neuron development, immunity, and cancer cell metastasis.

The matricellular protein CCN1 in tissue injury repair.

The expression of Ccn1 (Cyr61) is essential for cardiovascular development during embryogenesis, whereas in adulthood it is associated with inflammation, wound healing, injury repair, and related pathologies including fibrosis and cancer. Recent studies have found that CCN1 plays a critical role in promoting wound healing and tissue repair. Mechanistically, CCN1 functions through direct interaction with specific integrin receptors expressed in various cell types in the wound tissue microenvironment to coordinate diverse cellular functions for repair. Here we briefly summarize the current knowledge on the functions of CCN1 in tissue injury repair and discuss pertinent unanswered questions.

Assessment of vascularity in hepatic tumors: comparison of power Doppler sonography and intraarterial CO(2)-enhanced sonography.

OBJECTIVE: The purpose of the study was to compare power Doppler sonography with intraarterial CO(2)-enhanced sonography for revealing vascularity in treated and untreated hepatic tumors. SUBJECTS AND METHODS: Fifty-five patients with 93 liver tumors were prospectively examined with power Doppler sonography and CO(2)-enhanced sonography. These tumors included 29 hepatocellular carcinomas in patients with no previous treatment, 26 treated hepatocellular carcinomas, and 38 hemangiomas. The vascular depiction of power Doppler sonography was compared with that obtained in the early phase of CO(2)-enhanced sonography. The results of angiography were also recorded for comparison. RESULTS: In the hepatocellular carcinomas, power Doppler sonography was the same as CO(2)-enhanced sonography in 18 (62%) of 29 tumors, was inferior to CO(2)-enhanced sonography in nine (31%) of 29 tumors, and was superior to CO(2)-enhanced sonography in two (7%) of 29 tumors. In the treated hepatocellular carcinomas, power Doppler sonography was the same as CO(2)-enhanced sonography in 15 (58%) of 26 tumors and was inferior in 11 (42%) of 26 tumors. In hemangiomas, the same vascularity was found in both studies in 15 (39%) of 38 tumors, CO(2)-enhanced sonography was superior in 22 (58%) of 38 tumors, and power Doppler sonography was superior in one (3%) of 38 tumors. As a whole, 45% of the 93 tumors showed better vascular depiction on CO(2)-enhanced sonography. However, 19.4% of tumors were hypovascular using power Doppler sonography but hypervascular using CO(2)-enhanced sonography. CONCLUSION: Power Doppler sonography is a useful technique for screening hepatic tumor vascularity. CO(2)-enhanced sonography is superior to power Doppler sonography in depicting tumor vascularity in treated hepatocellular carcinomas and in hemangiomas, especially small hemangiomas.