Caspase-resistant ROCK1 expression prolongs survival of Eµ-Myc B cell lymphoma mice.

Apoptosis is characterized by membrane blebbing and apoptotic body formation. Caspase cleavage of ROCK1 generates an active fragment that promotes actin-myosin-mediated contraction and membrane blebbing during apoptosis. Expression of caspase-resistant non-cleavable ROCK1 (Rock1 NC) prolonged survival of mice that rapidly develop B cell lymphomas due to Eµ-Myc transgene expression. Eµ-Myc; Rock1 NC mice had significantly fewer bone marrow cells relative to those in Eµ-Myc mice expressing wild-type ROCK1 (Rock1 WT), which was associated with altered cell cycle profiles. Circulating macrophage numbers were lower in Eµ-Myc; Rock1 NC mice, but there were higher levels of bone marrow macrophages, consistent with spontaneous cell death in Eµ-Myc; Rock1 NC mouse bone marrows being more inflammatory. Rock1 WT recipient mice transplanted with pre-neoplastic Eµ-Myc; Rock1 NC bone marrow cells survived longer than mice transplanted with Eµ-Myc; Rock1 WT cells, indicating that the survival benefit was intrinsic to the Eµ-Myc; Rock1 NC bone marrow cells. The results suggest that the apoptotic death of Eµ-Myc; Rock1 NC cells generates a proliferation-suppressive microenvironment in bone marrows that reduces cell numbers and prolongs B cell lymphoma mouse survival.

Immunogenic Death of Hepatocellular Carcinoma Cells in Mice Expressing Caspase-Resistant ROCK1 Is Not Replicated by ROCK Inhibitors.

The morphological changes during apoptosis help facilitate "immunologically silent" cell death. Caspase cleavage of the ROCK1 kinase results in its activation, which drives the forceful contraction of apoptotic cells. We previously showed that when ROCK1 was mutated to render it caspase-resistant, there was greater liver damage and neutrophil recruitment after treatment with the hepatotoxin diethylnitrosamine (DEN). We now show that acute DEN-induced liver damage induced higher levels of pro-inflammatory cytokines/chemokines, indicative of immunogenic cell death (ICD), in mice expressing non-cleavable ROCK1 (ROCK1nc). Hepatocellular carcinoma (HCC) tumours in ROCK1nc mice had more neutrophils and CD8+ T cells relative to mice expressing wild-type ROCK1, indicating that spontaneous tumour cell death also was more immunogenic. Since ICD induction has been proposed to be tumour-suppressive, the effects of two distinct ROCK inhibitors on HCC tumours was examined. Both fasudil and AT13148 significantly decreased tumour numbers, areas and volumes, but neither resulted in greater numbers of neutrophils or CD8+ T cells to be recruited. In the context of acute DEN-induced liver damage, AT13148 inhibited the recruitment of dendritic, natural killer and CD8+ T cells to livers. These observations indicate that there is an important role for ROCK1 cleavage to limit immunogenic cell death, which was not replicated by systemic ROCK inhibitor administration. As a result, concomitant administration of ROCK inhibitors with cancer therapeutics would be unlikely to result in therapeutic benefit by inducing ICD to increase anti-tumour immune responses.

Characterization of full-length p53 aggregates and their kinetics of formation.

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.

Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses.

Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3ß or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.

Defective apoptotic cell contractility provokes sterile inflammation, leading to liver damage and tumour suppression.

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage-associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour-suppressive mechanism.

Review of Boundary Violations in Rhode Island, 2012-2018.

Boundary violations are serious occurrences that result in a breach of the physician-patient relationship. Boundary violations are regulated by the Rhode Island Board of Medical Licensure and Discipline (Board). A review of all disciplinary actions from 2012 through 2018 involving physicians found boundary violations relating to sexual misconduct the most common boundary violations. All disciplinary actions that pertained to boundary violations were isolated, and trends regarding gender, medical specialty, and outcome of action were assessed. Sexual boundary violations (n=15) represented 94% of all boundary violations during this time period. Psychiatrists (31.3%), internists (25%) and family medicine physicians (18.8%) were the most common specialties of those who had boundary issues. Loss of license occurred for all physicians who had sexual intercourse with a patient. Reinstatement of license was possible for some physicians after a comprehensive forensic psychiatry evaluation. Physicians are reminded of the ethical obligations we have to our patients and the profession and to maintain a professional relationship with their patients at all times. This type of professional misconduct is preventable and avoids injury to patients and to the medical profession. [Full article available at http://rimed.org/rimedicaljournal-2019-03.asp].

ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a KrasG12D/p53R172H mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three-dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK-induced genes that facilitate extracellular matrix remodeling, with greatest fold-changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13 MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three-dimensional contexts. Treatment of KrasG12D/p53R172H PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor-associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.

Rho-associated coiled-coil containing kinases (ROCK): structure, regulation, and functions.

Rho-associated coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase. (1)(-) (5) They belong to the AGC family of serine/threonine kinases (6) and play vital roles in facilitating actomyosin cytoskeleton contractility downstream of RhoA and RhoC activation. Since their discovery, ROCK kinases have been extensively studied, unveiling their manifold functions in processes including cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs have been identified, ROCK1 (also called ROCK I, ROKß, Rho-kinase ß, or p160ROCK) and ROCK2 (also known as ROCK II, ROKα, or Rho kinase), hereafter collectively referred to as ROCK. In this review, we will focus on the structure, regulation, and functions of ROCK.