br STAR Methods br Author Contributions br Introduction Hyal

STAR★Methods
Author Contributions
Introduction Hyaluronan, a major constituent of the extracellular matrix (ECM), is produced by hyaluronan synthases (HASs) and degraded by hyaluronidases (HYALs) (Vigetti et al., 2014). Physiologically, hyaluronan exists in a form with a molecular weight of >1,000 kDa, termed high-molecular-weight hyaluronan (HMW-HA), which exhibits anti-inflammatory, antiproliferative, and antiangiogenic actions by binding with its cellular receptors such as CD44 and RHAMM (receptor for hyaluronan-mediated motility) (Misra et al., 2015). Recently, an unexpected link between hyaluronan, longevity, and cancer was discovered. The naked mole rat is an extremely long-lived and cancer-resistant rodent (Gorbunova et al., 2014). Naked mole rat-derived fibroblasts are hypersensitive to contact inhibition: they cease proliferation as soon as a few cell-cell contacts have been initiated (Seluanov et al., 2009). The “early contact inhibition” indicates the existence of an extracellular substance(s) that can inform individual Mouse iPSC Chemical Reprogramming Cocktails Kit plus about population density prior to substantial cell-cell interaction. Furthermore, naked mole rat-derived fibroblasts resist oncogenic transformation. Both the early contact inhibition and oncogene resistance are attributable to the production and secretion of very high-molecular-weight hyaluronan (>6,000 kDa) from naked mole rat cells and its interaction with CD44 (Tian et al., 2013). The tumor suppressive Hippo pathway controls organ size by regulating cell proliferation and apoptosis during development (Yu et al., 2015). The core component of the mammalian Hippo pathway comprises a cascade of two serine/threonine kinases, MST (MST1 and MST2) and LATS (LATS1 and LATS2), sequential activation of which negatively regulates the activity of the pro-oncogenic transcriptional coactivators YAP and TAZ via phosphorylation (Avruch et al., 2012). Involvement of Hippo signaling in contact inhibition of cell proliferation is corroborated by the finding that YAP overexpression antagonizes contact inhibition, while dominant-negative YAP restores it (Gumbiner and Kim, 2014). Unlike other signaling pathways, however, the Hippo pathway does not possess a cognate extracellular ligand and receptor system. Instead, it is regulated by a variety of signaling events triggered by the interaction of cells with the cell-extracellular matrix (ECM), raising the idea that an ECM component(s) can serve as a Hippo signal ligand in contact inhibition of cell growth. In fact, several recent reports showed the involvement of hyaluronan and CD44 in Hippo signaling regulation, although the underlying mechanisms are not well understood (Xu et al., 2010, Tsuneki and Madri, 2016). While acting as a major hyaluronan receptor, CD44 is known to be a consistent marker of cancer stem cells in a variety of malignancies (Bourguignon et al., 2014). Also, CD44 expression is often upregulated in aggressive cancers (Udabage et al., 2005), and an abundance of hyaluronan is an indicator of poor prognosis in several types of cancers (Sironen et al., 2011). A clue to reconcile the opposing biological roles of CD44 and/or hyaluronan in terms of oncogenesis is related to the size of hyaluronan that binds to CD44. Under pathological conditions such as inflammation and tumorigenesis, HMW-HA is extensively degraded by elevated HYALs, especially HYAL2, to low-molecular-weight hyaluronan (LMW-HA) (<1,000 kDa) (Monslow et al., 2015, Litwiniuk et al., 2016). In stark contrast with HMW-HA, interaction of LMW-HA with CD44 provokes pro-oncogenic cellular actions (Misra et al., 2015, Bohaumilitzky et al., 2017). However, the molecular mechanisms by which HMW-HA and LMW-HA exert opposing biological actions via the same CD44 receptor remain largely unknown.
Results
Discussion This study demonstrated that, in the condition of a low cell density in which HMW-HA secreted from the sparsely distributed cells is not yet sufficiently deposited in the pericellular space, PAR1b is distributed to the cytoplasm, where it forms a complex with the Hippo core kinase MST and inhibits the MST kinase activity by phosphorylating the SARAH domain. It has recently been reported that homo- or heterodimerization of MST1/2 via the SARAH domain is critical for kinase activation (Rawat et al., 2016). The PAR1b-mediated phosphorylation of the SARAH domain may thus inactivate MST by hampering its dimerization. As cell density increases, pericellularly accumulated HMW-HA binds to the membrane receptor CD44 and thereby induces membrane clustering of CD44, resulting in recruitment of PAR1b by the CD44 intracellular domain. The CD44-PAR1b complex formation then disrupts and/or prevents formation of the Hippo signaling-inhibitory PAR1b-MST complex. Once liberated from the constraint by PAR1b, MST stimulates LATS to inhibit YAP, leading to HMW-HA-mediated growth inhibition at a high cell density, which contributes to contact inhibition. Under non-homeotic conditions such as inflammation and tumorigenesis, elevated HYAL2 degrades HMW-HA to LMW-HA (Monslow et al., 2015, Litwiniuk et al., 2016). The resultant LMW-HA binds to CD44 and competitively inhibits HMW-HA-mediated CD44 clustering, thereby preventing Hippo signaling activation by HMW-HA.