Functional Role of Kallikrein 6 in Regulating Immune Cell Survival

Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur.Using murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice.KLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis

Astrocyte heterogeneity across the brain and spinal cord occurs developmentally, in adulthood and in response to demyelination.

Astrocytes have emerged as essential regulators of function and response to injury in the brain and spinal cord, yet very little is known about regional differences that exist. Here we compare the expression of key astroglial markers (glial fibrillary acidic protein (GFAP) and Aldehyde Dehydrogenase-1 Family Member L1 (ALDH1L1)) across these disparate poles of the neuraxis, tracking their expression developmentally and in the context of demyelination. In addition, we document changes in the astrocyte regulatory cytokine interleukin 6 (IL-6), and its signaling partner signal transducer and activator of transcription 3 (STAT3), in vivo and in vitro. Results demonstrate that GFAP expression is higher in the developing and adult spinal cord relative to brain. Comparisons between GFAP and ALDH1L1 expression suggest elevations in spinal cord GFAP during the early postnatal period reflect an accelerated appearance of astrocytes, while elevations in adulthood reflect higher expression by individual astrocytes. Notably, increases in spinal cord compared to whole brain GFAP were paralleled by higher levels of IL-6 and STAT3. Equivalent elevations in GFAP, GFAP/ALDH1L1 ratios, and in IL-6, were observed in primary astrocyte cultures derived from spinal cord compared to cortex. Also, higher levels of GFAP were observed in the spinal cord compared to the brain after focal demyelinating injury. Altogether, these studies point to key differences in astrocyte abundance and the expression of GFAP and IL-6 across the brain and spinal cord that are positioned to influence regional specialization developmentally and responses occurring in the context of injury and disease

The autolytic regulation of human kallikrein-related peptidase 6

Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS) lesions. Inhibition of KLK6 results in delayed onset and reduced severity of symptoms associated with experimental autoimmune encephalomyelitis, suggesting a key effector role for this protease in CNS inflammatory disease. KLK6 has been shown to autolytically cleave internally, leading to inactivation and suggesting a negative feedback inhibition control mechanism. Alternatively, the ability of KLK6 to self-activate has also been reported, suggesting a positive feedback activation loop control mechanism. Activation of pro-KLK6 requires hydrolysis after a Lys residue; however, KLK6 exhibits 2 order of magnitude reduced affinity for hydrolysis after Lys versus Arg residues; therefore, the ability to autolytically activate has been called into question. in the present study the catalytic activity of KLK6 toward its pro-sequence and internal autolytic sequence is characterized. the results show that the ability of KLK6 to activate pro-KLK6 is essentially negligible when compared to the rate of the internal autolytic inactivation or to the ability of other proteases to activate pro-KLK6. the results thus show that the primary autolytic regulatory mechanism of KLK6 is negative feedback inhibition, and activation is likely achieved through the action of a separate protease.Florida State Univ, Coll Med, Dept Biomed Sci, Tallahassee, FL 32306 USAFlorida State Univ, Coll Med, Dept Chem & Biochem, Tallahassee, FL 32306 USAMayo Med & Grad Sch, Program Mol Neurosci, Rochester, MN 55905 USAMayo Med & Grad Sch, Dept Neurol, Rochester, MN 55905 USAMayo Med & Grad Sch, Dept Phys Med & Rehabil, Rochester, MN 55905 USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Increases in GFAP after demyelinating injury are greater in the spinal cord compared to brain.

<p>Photomicrographs show immunoreactivity for GFAP or ALDH1L1 in (A) the corpus callosum, or (B) the dorsal column white matter of adult mice at base line, and at 14 d after microinjection of the demyelinating agent lysolecithin. Histograms show the percent area of GFAP immunofluorescence, and expression of GFAP/ALDH1L1+ astrocyte, was significantly greater in the spinal cord compared to the corpus callosum 14d post-lysolecithin lesion. (*P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, Students t-test), Scale bar = 50μm.</p