Background/Purpose: There is an urgent unmet need for effective therapeutic approaches for Systemic Sclerosis (SSc), a systemic autoimmune disease characterized by progressive fibrosis of skin and multiple internal organs and severe microvascular alterations. The molecular mechanisms responsible for the fibrotic process in SSc have not been fully identified, although numerous recent studies have demonstrated a crucial role of various protein kinases in the development of tissue fibrotic reactions. Here we examined the role of a novel group of kinases in the activation of fibrotic phenotype in SSc dermal fibroblasts.

PIM (Proviral Integration Moloney virus) family of serine/threonine protein kinases consists of three members PIM1, PIM2 and PIM3 that promote growth factor-independent proliferation of numerous cell types by phosphorylating a range of cellular proteins. Both PIM1 and PIM2 kinase are involved in the control of cell growth, differentiation and apoptosis. PIM kinases do not have an identified regulatory domain, therefore once transcribed they are constitutively active. Recent crystallography studies revealed that, unlike other kinases, PIM1 and PIM2 possess a hinge region which creates a unique binding pocket for ATP, thus offering a target for an increasing number of potent small-molecule PIM kinase inhibitors such as the novel PIM1/2 Kinase inhibitor VI we studied here.

Methods: PIM1 levels were assessed by Western blot analysis of cell lysates from confluent dermal fibroblasts obtained from skin biopsies from the leading edge of forearm lesions of patients with diffuse SSc of recent onset and from healthy donors. SSc dermal fibroblasts were grown in culture and treated with the PIM1/2 kinase inhibitor. Western blot analysis of cell lysates were performed to examine the effects of the PIM1/2 inhibitor employing specific antibodies for PIM1, αSMA, and housekeeping protein. Media from cell cultures were collected and Western blots for collagen type I and fibronectin were performed. Activation of PIM1 endogenous gene expression in normal fibroblasts was performed employing lentiviral CRISPR activation plasmid particles. Control cells were transduced with control lentiviral particles.

Results: SSc dermal fibroblasts displayed marked elevation of PIM1 levels in comparison with normal fibroblasts ( >2-Fold). PIM1/2 kinase inhibitor treatment of cultured SSc dermal fibroblasts did not cause morphological changes or detectable cytotoxicity at the concentrations employed, however, the levels of type I collagen and fibronectin production were markedly decreased as a result of PIM1/2 activity inhibition. Lentiviral overexpression of PIM1 in normal dermal fibroblasts resulted in a remarkable upregulation of the expression and production of numerous profibrotic proteins including collagen type I, αSMA and fibronectin.

Conclusion: The results indicate that PIM1 plays an important role in the molecular mechanisms responsible for the exaggerated fibrotic process in SSc. Thus, inhibition of PIM kinases provides a novel therapeutic target for the development of potent antifibrotic drugs for SSc and other fibrotic disorders.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/induction-of-a-profibrotic-phenotype-in-normal-dermal-fibroblasts-by-expression-of-pim1-kinase-and-demonstration-of-antifibrotic-effects-of-inhibition-of-pim-kinases-in-systemic-sclerosis-dermal-fibro/