Pyrophosphate therapy prevents trauma-induced calcification in the mouse model of neurogenic heterotopic ossification.

Trauma-induced calcification is the pathological consequence of complex injuries which often affect the central nervous system and other parts of the body simultaneously. We demonstrated by an animal model recapitulating the calcification of the above condition that adrenaline transmits the stress signal of brain injury to the calcifying tissues. We have also found that although the level of plasma pyrophosphate, the endogenous inhibitor of calcification, was normal in calcifying animals, it could not counteract the acute calcification. However, externally added pyrophosphate inhibited calcification even when it was administered after the complex injuries. Our finding suggests a potentially powerful clinical intervention of calcification triggered by polytrauma injuries which has no effective treatment.

Protein phosphatase-1 is involved in the maintenance of normal homeostasis and in UVA irradiation-induced pathological alterations in HaCaT cells and in mouse skin.

The number of ultraviolet (UV) radiation-induced skin diseases such as melanomas is on the rise. The altered behavior of keratinocytes is often coupled with signaling events in which Ser/Thr specific protein kinases and phosphatases regulate various cellular functions. In the present study the role of protein phosphatase-1 (PP1) was investigated in the response of human keratinocyte (HaCaT) cells and mouse skin to UV radiation. PP1 catalytic subunit (PP1c) isoforms, PP1cα/γ and PP1cδ, are all localized to the cytoskeleton and cytosol of keratinocytes, but PP1cδ was found to be dominant over PP1α/γ in the nucleus. PP1c-silencing in HaCaT cells decreased the phosphatase activity and suppressed the viability of the cells. Exposure to a 10 J/cm(2) UVA dose induced HaCaT cell death and resulted in a 30% decrease of phosphatase activity. PP1c-silencing and UVA irradiation altered the gene expression profile of HaCaT cells and suggested that the expression of 19 genes was regulated by the combined treatments with many of these genes being involved in malignant transformation. Microarray analysis detected altered expression levels of genes coding for melanoma-associated proteins such as keratin 1/10, calcium binding protein S100A8 and histone 1b. Treatment of Balb/c mice with the PP1-specific inhibitor tautomycin (TM) exhibited increased levels of keratin 1/10 and S100A8, and a decreased level of histone 1b proteins following UVA irradiation. Moreover, TM treatment increased pigmentation of the skin which was even more apparent when TM was followed by UVA irradiation. Our data identify PP1 as a regulator of the normal homeostasis of keratinocytes and the UV-response.

Creation of the first monoclonal antibody recognizing an extracellular epitope of hABCC6.

Mutations in the ABCC6 gene result in calcification diseases such as pseudoxanthoma elasticum or Generalized Arterial Calcification of Infancy. Generation of antibodies recognizing an extracellular (EC) epitope of ABCC6 has been hampered by the short EC segments of the protein. To overcome this limitation, we immunized bovine FcRn transgenic mice exhibiting an augmented humoral immune response with Human Embryonic Kidney 293 cells cells expressing human ABCC6 (hABCC6). We obtained a monoclonal antibody recognizing an EC epitope of hABCC6 that we named mEChC6. Limited proteolysis revealed that the epitope is within a loop in the N-terminal half of ABCC6 and probably spans amino acids 338-347. mEChC6 recognizes hABCC6 in the liver of hABCC6 transgenic mice, verifying both specificity and EC binding to intact hepatocytes.

Myosin phosphatase accelerates cutaneous wound healing by regulating migration and differentiation of epidermal keratinocytes via Akt signaling pathway in human and murine skin.

Wound healing is a complex sequence of cellular and molecular processes such as inflammation, cell migration, proliferation and differentiation. ROCK is a widely investigated Ser/Thr kinase with important roles in rearranging the actomyosin cytoskeleton. ROCK inhibitors have already been approved to improve corneal endothelial wound healing. The purpose of this study was to investigate the functions of myosin phosphatase (MP or PPP1CB), a type-1 phospho-Ser/Thr-specific protein phosphatase (PP1), one of the counter enzymes of ROCK, in skin homeostasis and wound healing. To confirm our hypotheses, we applied tautomycin (TM), a selective PP1 inhibitor, on murine skin that caused the arrest of wound closure. TM suppressed scratch closure of HaCaT human keratinocytes without having influence on the survival of the cells. Silencing of, the regulatory subunit of MP (MYPT1 or PPP1R12A), had a negative impact on the migration of keratinocytes and it influenced the cell-cell adhesion properties by decreasing the impedance of HaCaT cells. We assume that MP differentially activates migration and differentiation of keratinocytes and plays a key role in the downregulation of transglutaminase-1 in lower layers of skin where no differentiation is required. MAPK Proteome Profiler analysis on human ex vivo biopsies with MYPT1-silencing indicated that MP contributes to the mediation of wound healing by regulating the Akt signaling pathway. Our findings suggest that MP plays a role in the maintenance of normal homeostasis of skin and the process of wound healing.

Functional Rescue of ABCC6 Deficiency by 4-Phenylbutyrate Therapy Reduces Dystrophic Calcification in Abcc6-/- Mice.

Soft-tissue calcification is associated with aging, common conditions such as diabetes or hypercholesterolemia, and with certain genetic disorders. ABCC6 is an efflux transporter primarily expressed in liver facilitating the release of adenosine triphosphate from hepatocytes. Within the liver vasculature, adenosine triphosphate is converted into pyrophosphate, a major inhibitor of ectopic calcification. ABCC6 mutations thus lead to reduced plasma pyrophosphate levels, resulting in the calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Most mutations in ABCC6 are missense, and many preserve transport activity but are retained intracellularly. We have previously shown that the chemical chaperone 4-phenylbutyrate (4-PBA) promotes the maturation of ABCC6 mutants to the plasma membrane. In a humanized mouse model of pseudoxanthoma elasticum, we investigated whether 4-PBA treatments could rescue the calcification inhibition potential of selected ABCC6 mutants. We used the dystrophic cardiac calcification phenotype of Abcc6-/- mice as an indicator of ABCC6 function to quantify the effect of 4-PBA on human ABCC6 mutants transiently expressed in the liver. We showed that 4-PBA administrations restored the physiological function of ABCC6 mutants, resulting in enhanced calcification inhibition. This study identifies 4-PBA treatment as a promising strategy for allele-specific therapy of ABCC6-associated calcification disorders.

Oral administration of pyrophosphate inhibits connective tissue calcification.

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1, respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1-/- offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low-cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification.

Inhibition of protein phosphatase-1 and -2A decreases the chemosensitivity of leukemic cells to chemotherapeutic drugs.

The phosphorylation of key proteins balanced by protein kinases and phosphatases are implicated in the regulation of cell cycle and apoptosis of malignant cells and influences anticancer drug actions. The efficacy of daunorubicin (DNR) in suppression of leukemic cell survival was investigated in the presence of tautomycin (TM) and calyculin A (CLA), specific membrane permeable inhibitors of protein phosphatase-1 (PP1) and -2A (PP2A), respectively. CLA (50 nM) or TM (1µM) suppressed viability of THP-1 and KG-1 myeloid leukemia cell lines to moderate extents; however, they significantly increased survival upon DNR-induced cell death. CLA increased the phosphorylation level of Erk1/2 and PKB/Akt kinases, the retinoblastoma protein (pRb), decreased caspase-3 activation by DNR and increased the phosphorylation level of the inhibitory sites (Thr696 and Thr853) in the myosin phosphatase (MP) target subunit (MYPT1) as well as in a 25kDa kinase-enhanced phosphatase inhibitor (KEPI)-like protein. TM induced enhanced phosphorylation of pRb only, suggesting that this event may be a common factor upon CLA-induced PP2A and TM-induced PP1 inhibitory influences on cell survival. Silencing PP1 by siRNA in HeLa cells, or overexpression of Flag-KEPI in MCF-7 cells coupled with inducing its phosphorylation by PMA or CLA, resulted in increased phosphorylation of pRb. Our results indicate that PP1 directly dephosphorylates pRb, while PP2A might have an indirect influence via mediating the phosphorylation level of PP1 inhibitory proteins. These data imply the importance of PP1 inhibitory proteins in controlling the phosphorylation state of key proteins and regulating drug sensitivity and apoptosis in leukemic cells.

Identification of protein phosphatase interacting proteins from normal and UVA-irradiated HaCaT cell lysates by surface plasmon resonance based binding technique using biotin-microcystin-LR as phosphatase capturing molecule.

Identification of the interacting proteins of protein phosphatases is crucial to understand the cellular roles of these enzymes. Microcystin-LR (MC-LR), a potent inhibitor of protein phosphatase-1 (PP1), -2A (PP2A), PP4, PP5 and PP6, was biotinylated, immobilized to streptavidin-coupled sensorchip surface and used in surface plasmon resonance (SPR) based binding experiments to isolate phosphatase binding proteins. Biotin-MC-LR captured PP1 catalytic subunit (PP1c) stably and the biotin-MC-LR-PP1c complex was able to further interact with the regulatory subunit (MYPT1) of myosin phosphatase. Increased biotin-MC-LR coated sensorchip surface in the Surface Prep unit of Biacore 3000 captured PP1c, PP2Ac and their regulatory proteins including MYPT1, MYPT family TIMAP, inhibitor-2 as well as PP2A-A and -Bα-subunits from normal and UVA-irradiated HaCaT cell lysates as revealed by dot blot analysis of the recovered proteins. Biotin-MC-LR was used for the subcellular localization of protein phosphatases in HaCaT cells by identification of phosphatase-bound biotin-MC-LR with fluorescent streptavidin conjugates. Partial colocalization of the biotin-MC-LR signals with those obtained using anti-PP1c and anti-PP2Ac antibodies was apparent as judged by confocal microscopy. Our results imply that biotin-MC-LR is a suitable capture molecule in SPR for isolation of protein phosphatase interacting proteins from cell lysates in sufficient amounts for immunological detection.