Breakdown of Phospholipid Asymmetry Triggers ADAM17-Mediated Rescue Events in Cells Undergoing Apoptosis.

ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through the cleavage of transmembrane substrates, including epidermal growth factor receptor (EGFR) ligands such as transforming growth factor (TGF)-alpha and Epiregulin (EREG). Several ADAM17 substrates are relevant to oncogenesis and tumor growth. We have presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. The scramblase Xkr8 is instrumental for calcium-independent exposure of PS in apoptotic cells. Xkr8 can be dually activated by caspase-3 and by kinases. In this investigation, we examined whether Xkr8 would modulate ADAM17 activity under apoptotic and non-apoptotic conditions. Overexpression of Xkr8 in HEK293T cells led to significantly increased caspase-dependent as well as PMA-induced release of EREG and TGF-alpha. Conversely, siRNA-mediated downregulation of Xkr8 in colorectal Caco-2 cancer cells led to decreased PS externalization upon induction of apoptosis, which was accompanied by reduced shedding of endogenously expressed EREG and reduced cell survival. We conclude that Xkr8 shares with conventional scramblases the propensity to upmodulate the ADAM-sheddase function. Liberation of growth factors could serve a rescue function in cells on the pathway to apoptotic death.

Influence of Anoctamin-4 and -9 on ADAM10 and ADAM17 Sheddase Function.

Ca2+-activated Cl- channels (TMEM16, also known as anoctamins) perform important functions in cell physiology, including modulation of cell proliferation and cancer growth. Many members, including TMEM16F/ANO6, additionally act as Ca2+-activated phospholipid scramblases. We recently presented evidence that ANO6-dependent surface exposure of phosphatidylserine (PS) is pivotal for the disintegrin-like metalloproteases ADAM10 and ADAM17 to exert their sheddase function. Here, we compared the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, and 10) on ADAM sheddase activity. Similar to ANO6, overexpression of ANO4 and ANO9 led to increased release of ADAM10 and ADAM17 substrates, such as betacellulin, TGFα, and amphiregulin (AREG), upon ionophore stimulation in HEK cells. Inhibitor experiments indicated that ANO4/ANO9-mediated enhancement of TGFα-cleavage broadened the spectrum of participating metalloproteinases. Annexin V-staining demonstrated increased externalisation of PS in ANO4/ANO9-overexpressing cells. Competition experiments with the soluble PS-headgroup phosphorylserine indicated that the ANO4/ANO9 effects were due to increased PS exposure. Overexpression of ANO4 or ANO9 in human cervical cancer cells (HeLa), enhanced constitutive shedding of the growth factor AREG and increased cell proliferation. We conclude that ANO4 and ANO9, by virtue of their scramblase activity, may play a role as important regulators of ADAM-dependent cellular functions.

Scramblases as Regulators of Proteolytic ADAM Function.

Proteolytic ectodomain release is a key mechanism for regulating the function of many cell surface proteins. The sheddases ADAM10 and ADAM17 are the best-characterized members of the family of transmembrane disintegrin-like metalloproteinase. Constitutive proteolytic activities are low but can be abruptly upregulated via inside-out signaling triggered by diverse activating events. Emerging evidence indicates that the plasma membrane itself must be assigned a dominant role in upregulation of sheddase function. Data are discussed that tentatively identify phospholipid scramblases as central players during these events. We propose that scramblase-dependent externalization of the negatively charged phospholipid phosphatidylserine (PS) plays an important role in the final activation step of ADAM10 and ADAM17. In this manuscript, we summarize the current knowledge on the interplay of cell membrane changes, PS exposure, and proteolytic activity of transmembrane proteases as well as the potential consequences in the context of immune response, infection, and cancer. The novel concept that scramblases regulate the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface.

Role of ADAM10 and ADAM17 in Regulating CD137 Function.

Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.

Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 activity and the ERK signalling pathway.

Human papillomaviruses (HPV) are causative agents of various tumours such as cervical cancer. HPV binding to the cell surface of keratinocytes leads to virus endocytosis at tetraspanin enriched microdomains. Complex interactions of the capsid proteins with host proteins as well as ADAM17-dependent ERK1/2 signal transduction enable the entry platform assembly of the oncogenic HPV type 16. Here, we studied the importance of tetraspanin CD9, also known as TSPAN29, in HPV16 infection of different epithelial cells. We found that both overexpression and loss of the tetraspanin decreased infection rates in cells with low endogenous CD9 levels, while reduction of CD9 expression in keratinocytes that exhibit high-CD9 protein amounts, led to an increase of infection. Therefore, we concluded that low-CD9 supports infection. Moreover, we found that changes in CD9 amounts affect the shedding of the ADAM17 substrate transforming growth factor alpha (TGFα) and the downstream phosphorylation of ERK. These effects correlate with those on infection rates suggesting that a specific CD9 optimum promotes ADAM17 activity, ERK signalling and virus infection. Together, our findings implicate that CD9 regulates HPV16 infection through the modulation of ADAM17 sheddase activity.

Extracellular sphingomyelinase activity impairs TNF-α-induced endothelial cell death via ADAM17 activation and TNF receptor 1 shedding.

ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, is an important regulator of endothelial cell proliferation and cell survival. The protease controls vital cellular functions through cleavage of growth factors, cytokines and their receptors including transforming growth factor-alpha (TGF-α), tumor necrosis factor-alpha (TNF-α) and TNF-α receptor 1 (TNFR1). TNF-α is the major inducer of endothelial cell death in cardiovascular diseases. The latter are also characterized by elevated plasma and tissue levels of extracellular sphingomyelinase (SMase). Whether the SMase affects ADAM activity and thus endothelial cell function has not been addressed to date. Here, we analyzed the effect of SMase on ADAM17-mediated shedding in COS7 cells and in human umbilical vein endothelial cells (HUVECs). Exposure to SMase significantly increased ADAM17-mediated release of alkaline-phosphatase (AP)-tagged TGF-α in COS7 cells and shedding of endogenously expressed TNFR1 in HUVECs. We previously presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase function. We found that SMase treatment led to PS externalization in both cell types. Transient non-apoptotic PS exposure is often mediated by Ca2+-dependent phospholipid scramblases. Accordingly, the Ca2+-chelator EGTA markedly reduced the breakdown of phospholipid asymmetry and shedding of TGF-α and TNFR1. Moreover, sheddase activity was significantly diminished in the presence of the competing PS-headgroup OPLS. SMase-stimulated TNFR1 shedding strikingly diminished TNF-α-induced signalling cascades and endothelial cell death. Taken together, our data suggest that SMase activity might act as protective factor for endothelial cells in cardiovascular diseases.

SPINK9 stimulates metalloprotease/EGFR-dependent keratinocyte migration via purinergic receptor activation.

Serine protease inhibitors of the Kazal-type 9 (SPINK9) is a keratinocyte-derived cationic peptide that is found most abundantly in the upper layers of the palmar-plantar epidermis. In vitro, the peptide displays the capacity to inhibit specifically kallikrein-related peptidase 5 (KLK5). Here, we report that cells expressing SPINK9 secrete the peptide constitutively. Recombinant SPINK9 (rSPINK9) provoked transactivation of the EGFR in human keratinocytes, resulting in efficient downstream triggering of cell migration. Transactivation occurred via functional upregulation of a disintegrin and metalloproteases (ADAMs), as evidenced by suppression with a metalloproteinase inhibitor and an EGFR-blocking antibody. SPINK9 preparations isolated from human skin also displayed EGFR-transactivating capacity. The classical purinergic receptor antagonists oxidized ATP and pyridoxalphosphate-6-azophenyl-2',4',-disulfonic acid effectively suppressed EGFR transactivation by rSPINK9, indicating that in analogy to what has recently been reported for the cationic antimicrobial peptides cathelicidin LL-37 and bee venom melittin, purinergic receptors have an essential bridging role in promoting the upregulation of ADAM function by the cationic peptide. SPINK9 could represent an example of how a cationic peptide may subserve multiple and interrelated functions that contribute to the maintenance of the physical and immunological barrier of the skin.

Characterization of Spink6 in mouse skin: the conserved inhibitor of kallikrein-related peptidases is reduced by barrier injury.

The proteolytic regulation of the desquamation process by kallikrein-related peptidases (KLKs) is crucial for epidermal barrier function, and elevated KLK levels have been reported in atopic dermatitis. KLKs are controlled by specific inhibitors of the serine protease inhibitor of Kazal-type (Spink) family. Recently, SPINK6 was shown to be present in human stratum corneum. In order to investigate its role in epidermal barrier function, we studied mouse Spink6. Sequence alignment revealed that the Kazal domain of Spink6 is highly conserved in animals. Recombinant Spink6 efficiently inhibited mouse Klk5 and human KLK2, KLK4, KLK5, KLK6, KLK7, KLK12, KLK13, and KLK14, whereas human KLK1 and KLK8 were not inhibited. Spink6 was expressed in mouse epidermis mainly in the stratum granulosum, and the inner root sheath of hair follicles. Stimulation with flagellin, EGF, and IL-1ß did not alter Spink6 expression, whereas stimulation with tumor necrosis factor-α (TNFα)/IFNγ and all-trans retinoic acid resulted in a significant downregulation of Spink6 expression in cultured primary mouse keratinocytes. Mechanically and metabolically induced skin barrier dysfunction resulted both in a downregulation of Spink6 expression. Our study indicates that Spink6 is a potent inhibitor of KLKs and involved in skin barrier function.

Expression and regulation of murine SPINK12, a potential orthologue of human LEKTI2.

A balanced proteolytic activity in the epidermis is vital to maintain epidermal homoeostasis and barrier function. Distinct protease-inhibitor systems are operating in different epidermal layers. In the uppermost layer, the stratum corneum, kallikrein-like proteases and their inhibitors are responsible for desquamation of the cornified keratinocytes, thus regulating the integrity of the epidermal barrier. Following discovery and characterisation of the human multidomain inhibitor LEKTI (lympho-epithelial Kazal-type-related inhibitor, encoded by hspink5), several new members of the Kazal-type inhibitor family have been identified. Here we describe expression and regulation of murine SPINK12, a potential orthologue of human LEKTI2. Its expression was analysed by RT-PCR and immunohistochemistry revealing organ-specific pattern with high level of expression in the epidermis and several epithelia including the stomach, kidney and uterus. In addition, mSPINK12 expression in the epidermis of skin at footpads, where stratification is markedly pronounced, was several folds higher than in the abdominal epidermis. mSPINK12 mRNA levels were not affected by any cytokines tested while treatment of primary murine keratinocytes with the combination of calcium and sorbitol resulted in a strong increase in its mRNA. It appears that mspink12 is especially expressed in the epidermal areas with thick skin and that its regulation generally responds to differentiation signals. mrSPINK12 shows an inhibitory activity against murine keratinocyte-derived trypsin-like proteolytic activity, thus, the protein does appear orthologous to human LEKTI2 and may play an role in the regulation of epithelial cell functions.